Pietro Speziale

Biofilm formation in Staphylococcus implant infections. A review of molecular mechanisms and implications for biofilm-resistant materials

Implant infections in orthopaedics, as well as in many other medical fields, are chiefly caused by staphylococci. The ability of growing within a biofilm enhances the chances of staphylococci to protect themselves from host defences, antibiotic therapies, and biocides. Advances in scientific knowledge on structural molecules (exopolysaccharide, proteins, teichoic acids, and the most recently described extracellular DNA), on the synthesis and genetics of staphylococcal biofilms, and on the complex network of signal factors that intervene in their control are here presented, also reporting on the emerging strategies to disrupt or inhibit them. The attitude of polymorphonuclear neutrophils and macrophages to infiltrate and phagocytise biofilms, as well as the ambiguous behaviour exhibited by these innate immune cells in biofilm-related implant infections, are here discussed. Research on anti-biofilm biomaterials is focused, reviewing materials loaded with antibacterial substances, or coated with anti-adhesive/anti-bacterial immobilized agents, or surfaced with nanostructures. Latter approaches appear promising, since they avoid the spread of antibacterial substances in the neighbouring tissues with the consequent risk of inducing bacterial resistance.

Role of fibronectin-binding MSCRAMMs in bacterial adherence and entry into mammalian cells

Most bacterial infections are initiated by the adherence of microorganisms to host tissues. This process involves the interaction of specific bacterial surface structures, called adhesins, with host components. In this review, we discuss a group of microbial adhesins known as Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) which recognize and bind FN. The interaction of bacteria with FN is believed to contribute significantly to the virulence of a number of microorganisms, including staphylococci and streptococci. Several FN-binding MSCRAMMs of staphylococci and streptococci exhibit a similar structural organization and mechanism of ligand recognition. The ligand-binding domain consists of tandem repeats of a approximately 45 amino acid long unit which bind to the 29-kDa N-terminal region of FN. The binding mechanism is unusual in that the repeat units are unstructured and appear to undergo a conformational change upon ligand binding. Apart from supporting bacterial adherence, FN is also involved in bacterial entry into non-phagocytic mammalian cells. A sandwich model has been proposed in which FN forms a molecular bridge between MSCRAMMs on the bacterial surface and integrins on the host cell. However, the precise mechanism of bacterial invasion and the roles of FN and integrins in this process have yet to be fully elucidated.

Antibiotic-loaded biomaterials and the risks for the spread of antibiotic resistance following their prophylactic and therapeutic clinical use.

Antibiotic-loaded biomaterials are currently part of standard medical procedures for both local treatment and prevention of implant infections. The achievement of local delivery of significant quantities of active drugs directly at the site of infection, bypassing or reducing the risks of systemic effects, represents a strong point in favor of this approach. When the aim is to resolve an existing infection, controlled local release of antibiotics can be properly targeted based on the characteristics of the bacterial isolate obtained from the infection site. Under these circumstances the choice of the antibiotic is rational and this local administration route offers new unprecedented possibilities for an efficacious in situ treatment, avoiding the adverse effects of conventional systemic chemotherapies. Although the idea of self sterilizing implants is appealing, controversial is the use of antibiotic-loaded biomaterials in uninfected tissues to prevent implant infections. Systems designed for prolonged release of prophylactic inhibitory or subinhibitory amounts of antibiotics, in absence of strict harmonized guidelines, raise concerns for their still weakly proved efficacy but, even more, for their possible contribution to enhancing biofilm formation and selecting resistant mutants. This consideration holds especially true if the antibiotic-loaded represents the first-line treatment against multiresistant strains.

Scenery of Staphylococcus implant infections in orthopedics

Infection is still the major complication of orthopedic implants and projections based on the actual trend indicate that total hip and knee arthroplasties and their consequent infection burden are destined to greatly increase. Staphylococcus aureus and Staphylococcus epidermidis are the leading etiologic agents of orthopedic implant infection. Here we report on epidemiology of implant-related Staphylococcus infections in orthopedics, also referring to our experience, and focus on the crucial role of bacterial adhesins and on their ability to direct the pathogenesis process. Bacteria initiate implant infection by adhering to biomaterials. In the early steps of infection, adhesins mediate the specific interaction between microbial cells and the extracellular matrix proteins filming biomaterial surface. Then adhesin-mediated anchorage allows bacteria to cling to the biomaterial surface and to produce a biofilm that favors their ability to resist antibiotics. With the aim to prevent implant-related infections, anti-infective and infection-resistant biomaterials are being developed. The research for novel therapeutic strategies is incited by the emergence of antibiotic-resistant bacteria. Vaccines against the adhesins or antisense molecules against virulence genes can open a future in combating implant infections.

A collagen receptor on Staphylococcus aureus strains isolated from patients with septic arthritis mediates adhesion to cartilage

Staphylococcus aureus strains isolated from patients with septic arthritis or osteomyelitis possess a collagen receptor present in two forms, which contains either two or three copies of a 187‐amino‐acid repeat motif. Collagen receptor‐positive strains adhered to both collagen substrata and cartilage in a time‐dependent process. Collagen receptor‐specific antibodies blocked bacterial adherence, as did preincubation of the substrate with a recombinant form of the receptor protein. Furthermore, polystyrene beads coated with the collagen receptor bound collagen and attached to cartilage. Taken together, these results suggest that the collagen receptor is both necessary and sufficient to mediate bacterial adherence to cartilage in a process that constitutes an important part of the pathogenic mechanism in septic arthritis.

Fibronectin-binding proteins of Staphylococcus aureus mediate activation of human platelets via fibrinogen and fibronectin bridges to integrin GPIIb/IIIa and IgG binding to the FcgammaRIIa receptor.

Staphylococcus aureus is a leading cause of infective endocarditis (IE). Platelet activation promoted by S. aureus resulting in aggregation and thrombus formation is an important step in the pathogenesis of IE. Here, we report that the fibrinogen/fibronectin‐binding proteins FnBPA and FnBPB are major platelet‐activating factors on the surface of S. aureus from the exponential phase of growth. Truncated derivatives of FnBPA, presenting either the fibrinogen‐binding A domain or the fibronectin‐binding BCD region, each promoted platelet activation when expressed on the surface of S. aureus or Lactococcus lactis, indicating two distinct mechanisms of activation. FnBPA‐promoted platelet activation is mediated by fibrinogen and fibronectin bridges between the A domain and the BCD domains, respectively, to the low affinity form of the integrin GPIIb/IIIa on resting platelets. Antibodies recognizing the FnBPA A domain or the complex between the FnBPA BCD domains and fibronectin were essential for activation promoted by bacteria expressing the A domain or the BCD domain respectively. Activation was inhibited by a monoclonal antibody (IV‐3) specific for the FcγRIIa IgG receptor on platelets. We propose that the activation of quiescent platelets by bacteria expressing FnBPs involves the formation of a bridge between the bacterial cell and the platelet surface by (i) fibronectin and fibrinogen interacting with the low affinity form of GPIIb/IIIa and (ii) by antibodies specific to FnBPs that engage the platelet Fc receptor FcγRIIa. Platelet activation by S. aureus clinical IE isolates from both the exponential and stationary phases of growth was completely inhibited by monoclonal antibody IV‐3 suggesting that the IgG–FcγRIIa interaction is of fundamental importance for platelet activation mediated by this organism. This suggests new avenues for development of therapeutics against vascular infections.

Immunization with Staphylococcus aureus Clumping Factor B, a Major Determinant in Nasal Carriage, Reduces Nasal Colonization in a Murine Model

ABSTRACT Staphylococcus aureus is responsible for a wide range of infections, including soft tissue infections and potentially fatal bacteremias. The primary niche for S. aureus in humans is the nares, and nasal carriage is a documented risk factor for staphylococcal infection. Previous studies with rodent models of nasal colonization have implicated capsule and teichoic acid as staphylococcal surface factors that promote colonization. In this study, a mouse model of nasal colonization was utilized to demonstrate that S. aureus mutants that lack clumping factor A, collagen binding protein, fibronectin binding proteins A and B, polysaccharide intercellular adhesin, or the accessory gene regulator colonized as well as wild-type strains colonized. In contrast, mutants deficient in sortase A or clumping factor B (ClfB) showed reduced nasal colonization. Mice immunized intranasally with killed S. aureus cells showed reduced nasal colonization compared with control animals. Likewise, mice that were immunized systemically or intranasally with a recombinant vaccine composed of domain A of ClfB exhibited lower levels of colonization than control animals exhibited. A ClfB monoclonal antibody (MAb) inhibited S. aureus binding to mouse cytokeratin 10. Passive immunization of mice with this MAb resulted in reduced nasal colonization compared with the colonization observed after immunization with an isotype-matched control antibody. The mouse immunization studies demonstrate that ClfB is an attractive component for inclusion in a vaccine to reduce S. aureus nasal colonization in humans, which in turn may diminish the risk of staphylococcal infection. As targets for vaccine development and antimicrobial intervention are assessed, rodent nasal colonization models may be invaluable.

The serine-aspartate repeat (Sdr) protein family in Staphylococcus epidermidis.

Staphylococcus epidermidis can express three different cell-surface-associated proteins, designated SdrF, SdrG and SdrH, that contain serine-aspartate dipeptide repeats. Proteins SdrF and SdrG are similar in sequence and structural organization to the Sdr proteins of Staphylococcus aureus and comprise unique 625- and 548-residue A regions at their N termini, respectively, followed by 110-119-residue B-repeat regions and SD-repeat regions. The C termini contain LPXTG motifs and hydrophobic amino acid segments characteristic of surface proteins covalently anchored to peptidoglycan. In contrast, SdrH has a short 60-residue A region at its N terminus followed by a SD-repeat region, a unique 277-residue C region and a C-terminal hydrophobic segment. SdrH lacks a LPXTG motif. Recombinant proteins representing the A regions of SdrF, SdrG and SdrH were expressed and purified from Escherichia coli. Antisera specific to these proteins were raised in rabbits and used to identify Sdr proteins expressed by S. epidermidis. Only SdrF was released from lysostaphin-generated protoplasts of cells grown to late-exponential phase. SdrG and SdrH remained associated with the protoplast fraction and thus appear to be ineffectively sorted along the conventional pathway used for cell-wall-anchored proteins. In Southern hybridization analyses, the sdrG and sdrH genes were present in all 16 strains tested, whilst sdrF was present in 12 strains. Antisera from 16 patients who had recovered from S. epidermidis infections contained antibodies that reacted with recombinant A regions of SdrG and SdrH, suggesting that these proteins can be expressed during infection.

Toluidine Blue-Mediated Photodynamic Effects on Staphylococcal Biofilms

ABSTRACT Staphylococci are important causes of nosocomial and medical-device-related infections. Their virulence is attributed to the elaboration of biofilms that protect the organisms from immune system clearance and to increased resistance to phagocytosis and antibiotics. Photodynamic treatment (PDT) has been proposed as an alternative approach for the inactivation of bacteria in biofilms. In this study, we have investigated the effect of the photodynamic action of toluidine blue O (TBO) on the viability and structure of biofilms of Staphylococcus epidermidis and of a methicillin-resistant Staphylococcus aureus strain. Significant inactivation of cells was observed when staphylococcal biofilms were exposed to TBO and laser simultaneously. The effect was found to be light dose dependent. Confocal laser scanning microscopic study suggested damage to bacterial cell membranes in photodynamically treated biofilms. In addition, scanning electron microscopy provided direct evidence for the disruption of biofilm structure and a decrease in cell numbers in photodynamically treated biofilms. Furthermore, the treatment of biofilms with tetrasodium EDTA followed by PDT enhanced the photodynamic efficacy of TBO in S. epidermidis, but not in S. aureus, biofilms. The results suggest that photodynamic treatment may be a useful approach for the inactivation of staphylococcal biofilms adhering to solid surfaces of medical implants.

Extracellular DNA in biofilms.

Extracellular DNA (eDNA) is an important biofilm component that was recently discovered. Its presence has been initially observed in biofilms of Pseudomonas aeruginosa, Streptococcus intermedius, Streptococcus mutans, then Enterococcus faecalis and staphylococci. Autolysis is the common mechanism by which eDNA is released. In P. aeruginosa eDNA is generated by lysis of a bacterial subpopulation, under control of quorum sensing system. In E. faecalis autolysis proceeds in a fratricide mode, resulting from a process similar to necrosis of eukaryotic cells. In Staphylococcus aureus autolysis originates by an altruistic suicide, i.e., a programmed cell death similar to apoptosis of eukaryotic cells. In S. aureus autolysis is mediated by murein hydrolase, while in S. epidermidis by the autolysin protein AtlE. In P. aeruginosa eDNA is located primarily in the stalks of mushroom-shaped multicellular structures. In S. aureus the crucial role of eDNA in stabilizing biofilm is highlighted by the disgregating effect of DNase I. eDNA represents an important mechanism for horizontal gene transfer in bacteria. eDNA and other microbial structural motifs are recognized by the innate immune system via the TLR family of pattern recognition receptors (PRRs).