The presence of both bone sialoprotein-binding protein gene and collagen adhesin gene as a typical virulence trait of the major epidemic cluster in isolates from orthopedic implant infections
Introduction
Staphylococcus aureus is a major, highly clonal, pathogen causing implant infections in orthopaedics [1], [2]. The highly clonal nature of S. aureus, described by different authors in recent molecular epidemiology and comparative genomics studies [3], [4], points out to the versatility of this bacterium, which has evolved into a variety of divergent strain types. Under selective pressure, S. aureus clones have evolved developing distinct virulence potentials and, in some cases, great abilities to broadly spread becoming epidemic or even pandemic. Bacterial adhesion and subsequent colonization of host tissues represent an early critical step of the pathogenesis of S. aureus infections. S. aureus adhesins, also called MSCRAMMs, i.e. Microbial Surface Components Recognizing Adhesive Matrix Molecules [5], have been described to play an important role in various processes of infection pathogenesis such as tropism, invasion, intracellular penetration, and, in the peri-implant tissues, bacterial adhesion on biomaterials coated by host extracellular matrix (ECM) proteins. A number of adhesins involved in adhesion on indwelling devices appear multifunctional, and Fibronectin-binding proteins A and B (FnBPA and FnBPB) and Clumping factors A and B (ClfA and ClfB) [6], [7] are among those known to bind more than a specific ligand. For instance, FnBPA in addition to fibronectin can bind fibrinogen and elastin, and ClfA binds fibrin besides fibrinogen γ-chain. Binding of fibronectin by FnBPs was found to be crucial in the invasion of eukaryotic cells, where the ECM protein serves as a bridging molecule between the adhesin and the integrin α5β1 [8], [9], enabling the internalization of the bacteria within the cells. On the other hand, there are cases in which more adhesins recognize the same ECM ligand. For instance, in addition to Clfs, there is a number of other known fibrinogen-binding adhesins (FbBPs), including Fib and FbPA. The existence of many adhesins, some ones capable of binding the same target protein and exhibiting apparently overlapping functions, and others binding more than a single ECM component, makes it difficult to clear up and weigh their role in pathogenesis and to elucidate their complex interplay [9], [10], [11].
However, in spite of these difficulties, there is sufficient information for certain adhesins to attest that they significantly contribute to strain virulence and to the pathogenesis of specific types of infections. The collagen adhesin (CNA), encoded by the cna gene, is a virulence factor that has been shown to be important in the pathogenesis of various types of infections [12]. CNA mediates staphylococcal adhesion on cartilage tissue and has been found to play a role in orthopaedic implant infections [13], [14], septic arthritis, osteomyelitis, endocarditis, and keratitis [15], [16], [17], [18].
Other adhesins are far from being fully investigated. Elastin-binding protein of S. aureus (EbpS) has been identified as an adhesin that binds to soluble elastin or tropoelastin, but the structure and exact function of EbpS remain to be elucidated [19]. In spite of the limited knowledge available, EbpS and the laminin-binding adhesin encoded by the eno gene represent virulence factors generally found in the majority of S. aureus isolates.
An interesting group of MSCRAMMs members is represented by the Serine–Aspartate repeat (Sdr) proteins. In addition to the earlier mentioned Cfls, Sdr proteins include SdrC, SdrD, SdrE, and the bone sialoprotein-binding protein (Bbp). Sdr proteins have comparable structural organization, but they are not closely related, with only 20%–30% amino acid residues similarity [20]. Bbp is an allelic variant of SdrE and is the only of these adhesins whose ECM ligand has been identified. Bone sialoprotein, the binding target of Bbp, is an ECM highly glycosylated and sulphated phosphoprotein that is found almost exclusively in mineralized connective tissues [21], where it represents 10% of the non-collagenous proteins of the matrix, being mostly synthesized in osseous tissue under the surface of joint cartilage [22], [23] and dentine. In an in vivo animal model, bone sialoprotein-binding capacity was found in all staphyl-ococci producing septic arthritis [18], [24], [25], [26], this suggesting that Bbp could represent an important virulence factor.
The real functions of the other Sdr proteins, respectively SdrC, SdrD, and SdrE, are still to be clarified and, at now, their respective ligands have not yet been determined [20]. It has been documented that at least two of the genes encoding for these Sdr proteins are tandemly arrayed and generally present in the genome of S. aureus strains [27]. Moreover, in different studies, the gene sdrC, encoding for SdrC, resulted always present in all analyzed isolates [20], [26]. Conversely, the presence of the SdrE and its allelic variants was found to be highly correlated with staphylococcal invasiveness [28]. SdrD was more frequently observed in S. aureus isolates causing bone infections [29], while strains lacking both SdrD and SdrE were found to possess a decreased potential to infect bones [20].
In a previous study [3] we examined the antibiotic resistances and the toxigenicity of S. aureus strain types causing orthopaedic implant-related infections, in a collection of 200 S. aureus isolates, ribotyped by a RiboPrinter. The automated apparatus generates patterns based on restriction enzyme digestion and hybridization of the resulting fragments to the rDNA gene probes. Overall, multi-resistance was generally found to be associated to the largest epidemic strain types identified by automated ribotyping. Surprisingly, the most epidemic strain type was endowed with minimal antibiotic resistance and consisted of isolates free of the LukE/LuKD and the Panton–Valentine (PVL) leukocidins, pointing out to other undisclosed factors, different from the investigated antibiotic resistance determinants and leukocidins, as the source of its epidemic success. The present work aimed at further characterizing this epidemic clone, searching for some other virulence factors, which could be related to its successful spreading. All clinical isolates were analyzed for the presence of a panel of genes encoding for relevant adhesins. The obtained data served to draw a specific MSCRAMMs profile for each different strain type and to search if any of these putative virulence traits were common to the isolates of the largest and most successful clone.
Section snippets
Bacterial strains
The investigation was conducted on 200 S. aureus isolates from orthopaedic implant-related infections. The isolates, previously described in [3], belong to the CRA/LM strain collection and are stored in the ISO 9001:2000 certified biobank of the Research Unit on Implant Infections of the Rizzoli Orthopaedic Institute.
Identification and storage
Staphylococcal species identification was performed by Api-Staph test (bioMérieux, Marcy l'Etoile, France) and subsequently confirmed by riboprinting [3].
PCR detection of target genes
The detection of the genes
Presence of adhesins in prevalent strain types
Previous analyses performed by automated ribotyping enlightened the existence of 98 distinct ribogroups in the collection of 200 isolates [3]. The 17 largest ribogroups, corresponding to the most frequent strain types, enlisted over 53% of all isolates. Once again confirming the validity of automated ribotyping to finely ascertain distinct clonal entities within single bacterial species, the different identified ribogroups were associated to diverse, well defined, profiles of adhesin genes.
Discussion
Our findings clearly reaffirm that strain subtyping by riboprinting is useful for the identification of distinct staphylococcal clones. Interestingly, in this differing from other subtyping techniques targeting the sequence of highly conserved genes of the core genome, distinct ribopatterns appear to correspond to clonal entities finely characterized even by distinguishable profiles of accessory genes such as adhesins. A limitation of this technique could perhaps reside on the tendency to
Conclusion
Epidemic clones appear to pursue different survival strategies, where adhesins, when present, exhibit diverse importance as virulence factors. The evidence that the major epidemic cluster of S. aureus recognized in this collection of isolates is characterized by the presence of CNA and Bbp suggests that couples of adhesins may act synergistically in the onset and progression of bacterial colonization and infection. A practical message arising from the present study is that strategies for the
Acknowledgements
This work was partially supported by a grant of the Italian Ministry of Health (identification code RF-IOR-2006-349032) “Opportunistic bacteria in orthopaedic implant infections. New strategies of molecular and gene therapies” (National Coordinator Prof. C.R. Arciola).
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