Interactions of Neutrophils with the Polymeric Molecular Components of the Biofilm Matrix in the Context of Implant-Associated Bone and Joint Infections.

In the presence of orthopedic implants, opportunistic pathogens can easily colonize the biomaterial surfaces, forming protective biofilms. Life in biofilm is a central pathogenetic mechanism enabling bacteria to elude the host immune response and survive conventional medical treatments. The formation of mature biofilms is universally recognized as the main cause of septic prosthetic failures. Neutrophils are the first leukocytes to be recruited at the site of infection. They are highly efficient in detecting and killing planktonic bacteria. However, the interactions of these fundamental effector cells of the immune system with the biofilm matrix, which is the true interface of a biofilm with the host cells, have only recently started to be unveiled and are still to be fully understood. Biofilm matrix macromolecules consist of exopolysaccharides, proteins, lipids, teichoic acids, and the most recently described extracellular DNA. The latter can also be stolen from neutrophil extracellular traps (NETs) by bacteria, who use it to strengthen their biofilms. This paper aims to review the specific interactions that neutrophils develop when they physically encounter the matrix of a biofilm and come to interact with its polymeric molecular components.

Biofilms in Periprosthetic Orthopedic Infections Seen through the Eyes of Neutrophils: How Can We Help Neutrophils?

Despite advancements in our knowledge of neutrophil responses to planktonic bacteria during acute inflammation, much remains to be elucidated on how neutrophils deal with bacterial biofilms in implant infections. Further complexity transpires from the emerging findings on the role that biomaterials play in conditioning bacterial adhesion, the variety of biofilm matrices, and the insidious measures that biofilm bacteria devise against neutrophils. Thus, grasping the entirety of neutrophil-biofilm interactions occurring in periprosthetic tissues is a difficult goal. The bactericidal weapons of neutrophils consist of the following: ready-to-use antibacterial proteins and enzymes stored in granules; NADPH oxidase-derived reactive oxygen species (ROS); and net-like structures of DNA, histones, and granule proteins, which neutrophils extrude to extracellularly trap pathogens (the so-called NETs: an allusive acronym for "neutrophil extracellular traps"). Neutrophils are bactericidal (and therefore defensive) cells endowed with a rich offensive armamentarium through which, if frustrated in their attempts to engulf and phagocytose biofilms, they can trigger the destruction of periprosthetic bone. This study speculates on how neutrophils interact with biofilms in the dramatic scenario of implant infections, also considering the implications of this interaction in view of the design of new therapeutic strategies and functionalized biomaterials, to help neutrophils in their arduous task of managing biofilms.

Antibacterial Activity on Orthopedic Clinical Isolates and Cytotoxicity of the Antimicrobial Peptide Dadapin-1.

In orthopedic surgery, biomaterial-associated infections represent a complication of serious concern. Most promising strategies to prevent these infections currently rely on the use of anti-infective biomaterials. Desirably, in anti-infective biomaterials, the antibacterial properties should be achieved by doping, grafting, or coating the material surfaces with molecules that are alternative to conventional antibiotics and exhibit a potent and highly specific activity against bacteria, without altering the biocompatibility. Antimicrobial peptides (AMPs) are among the most interesting candidate molecules for this biomaterial functionalization. Here, the potential expressed by the recently discovered peptide Dadapin-1 was explored by assaying its MIC, MBIC and MBC on clinical strains of relevant bacterial species isolated from orthopedic infections and by assessing its cytotoxicity on the human osteoblast-like MG63 cells. When appropriately tested in diluted Mueller Hinton Broth II (MHB II), Dadapin-1 exhibited significant antibacterial properties. MIC values were in the range of 3.1-6.2 µM for the gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus warneri, and 12.4-24.9 µM for the gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. Interestingly, the peptide was found non-cytotoxic, with an IC50 exceeding the highest concentration tested of 179 µM. Overall, Dadapin-1 expresses considerable potential for future application in the production of anti-infective biomaterials.

Searching for Virulence Factors among Staphylococcus lugdunensis Isolates from Orthopedic Infections: Correlation of ß-hemolysin, hemolysin III, and slush Genes with Hemolytic Activity and Synergistic Hemolytic Activity.

Staphylococcus lugdunensis is an emerging high-virulent pathogen. Here, the presence and expression of virulence genes (icaA, fbl, vwbl, fbpA, slush A, B and C, and genes of the putative ß-hemolysin and hemolysin III) and the ability to induce synergistic hemolytic activity and hemolysis after 24, 48 and 72 h were investigated in a collection of twenty-two S. lugdunensis clinical isolates. The collection of isolates, mainly from implant orthopedic infections, had previously been grouped by ribotyping/dendrogram analysis and studied for biofilm matrices, biomasses and antibiotic resistances. Two isolates, constituting a unique small ribogroup sharing the same cluster, exhibited an amplicon size of the slush operon (S. lugdunensis synergistic hemolysin) which was shorter than the expected 977 bp. This outcome can predict the genetic lineage of the S. lugdunensis strains. One isolate (cra1342) presented two deletions: one of 90 bp in slush A and the other of 91 bp in slush B. Another isolate (N860314) showed a single 193 bp deletion, which encompassed part of the slush B terminal sequence and most of slush C. The isolate N860314 was devoid of hemolytic activity after 24 h, and the first consideration was that the deleted region deals with the coding of the active enzymatic site of the slush hemolysin. On the other hand, cra1342 and N860314 isolates with different slush deletions and with hemolytic activity after 24 and 48 h, respectively, could have replaced the hemolytic phenotype through other processes.

Synthesis, Crystal Structure, and Antibacterial Properties of Silver-Functionalized Low-Dimensional Layered Zirconium Phosphonates.

New insoluble layered zirconium phosphate carboxyaminophosphonates (ZPs), with the general formula Zr2(PO4)H5[(O3PCH2)2N(CH2)nCOO]2·mH2O (n = 3, 4, and 5), have been prepared and characterized. The crystal structure for n = 3 and 4 samples was determined ab initio from X-ray powder diffraction data. The structure for n = 3 was monoclinic in space group C2/c with the following unit cell parameters: a = 34.346(1) Å, b = 8.4930(2) Å, c = 9.0401(2) Å, and ß = 97.15(1)°. The structure for n = 4 was triclinic in space group P1̅ with the following unit cell parameters: a = 17.9803(9) Å, b = 8.6066(4) Å, c = 9.0478(3) Å, α = 90.466(3)°, ß = 94.910(4)°, and γ = 99.552(4)°. The two structures had the same connectivity as Zr phosphate glycine diphosphonate (n = 1), as previously reported. By intercalation of short amines, these layered compounds were exfoliated in single lamella or packets of a few lamellae, which formed colloidal dispersions in water. After a thorough characterization, the dispersed lamellae were functionalized with Ag nanoparticles, which were grown in situ on the surface of exfoliated lamellae. Finally, their antimicrobial activity was tested on several Gram-positive and Gram-negative bacteria. All of these systems were found to be active against the four pathogens most frequently isolated from orthopedic prosthetic infections and often causative of nosocomial infections. Interestingly, they were found to express powerful inhibitory activity even against bacterial strains exhibiting a relevant profile of antibiotic resistance such as Staphylococcus aureus ATCC 700699.

Comparison of Automated Ribotyping, spa Typing, and MLST in 108 Clinical Isolates of Staphylococcus aureus from Orthopedic Infections.

108 isolates of Staphylococcus aureus, belonging to six large ribogroups according to the automated Ribo-Printer® system, were studied with two highly used molecular methods for epidemiological studies, namely multi-locus sequence typing (MLST) and spa typing, followed by BURP and eBURST v3 analysis for clustering spa types and sequence (ST) types. The aim was to evaluate whether automated ribotyping could be considered a useful screening tool for identifying S. aureus genetic lineages with respect to spa typing and MLST. Clarifying the relationship of riboprinting with these typing methods and establishing whether ribogroups fit single clonal complexes were two main objectives. Further information on the genetic profile of the isolates was obtained from agr typing and the search for the mecA, tst genes, and the IS256 insertion sequence. Automated ribotyping has been shown to predict spa clonal complexes and MLST clonal complexes. The high cost and lower discriminatory power of automated ribotyping compared to spa and MSLT typing could be an obstacle to fine genotyping analyzes, especially when high discriminatory power is required. On the other hand, numerous advantages such as automation, ease and speed of execution, stability, typeability and reproducibility make ribotyping a reliable method to be juxtaposed to gold standard methods.

Various biofilm matrices of the emerging pathogen Staphylococcus lugdunensis: exopolysaccharides, proteins, eDNA and their correlation with biofilm mass.

Staphylococcus lugdunensis is an emerging high-virulent pathogen causative of hospital-acquired infections. Biofilm formation is a complex pathogenic process that leads to well-established bacterial communities. There is a paucity of data on the composition of the biofilm matrix among S. lugdunensis strains. Here, twenty-two S. lugdunensis clinical isolates, mainly from orthopaedic infections but also from other clinical sources, were sub-grouped by ribotyping and dendrogram analysis. Biofilms were analysed by fluorimetric methods based on FITC-Wheat Germ Agglutinin, SYPRO Ruby and TOTO-1 dyes to detect exopolysaccharides, proteins and extracellular DNA (eDNA), respectively. Biofilm morphology was investigated under confocal laser scanning microscopy (CLSM). Isolates displayed intriguing diversities in biofilm mass and matrix composition. The content of exopolysaccharides was found to be to be strongly associated with the biofilm mass (R2 = 0.882), while the content of proteins turned out to be weakly (R2 = 0.465) and that of eDNA very weakly associated (R2 = 0.202) to the biofilm mass.

Emerging Issues and Initial Insights into Bacterial Biofilms: From Orthopedic Infection to Metabolomics.

Bacterial biofilms, enigmatic communities of microorganisms enclosed in an extracellular matrix, still represent an open challenge in many clinical contexts, including orthopedics, where biofilm-associated bone and joint infections remain the main cause of implant failure. This study explores the scenario of biofilm infections, with a focus on those related to orthopedic implants, highlighting recently emerged substantial aspects of the pathogenesis and their potential repercussions on the clinic, as well as the progress and gaps that still exist in the diagnostics and management of these infections. The classic mechanisms through which biofilms form and the more recently proposed new ones are depicted. The ways in which bacteria hide, become impenetrable to antibiotics, and evade the immune defenses, creating reservoirs of bacteria difficult to detect and reach, are delineated, such as bacterial dormancy within biofilms, entry into host cells, and penetration into bone canaliculi. New findings on biofilm formation with host components are presented. The article also delves into the emerging and critical concept of immunometabolism, a key function of immune cells that biofilm interferes with. The growing potential of biofilm metabolomics in the diagnosis and therapy of biofilm infections is highlighted, referring to the latest research.

Exploring the anticancer effects of standardized extracts of poplar-type propolis: In vitro cytotoxicity toward cancer and normal cell lines.

Propolis was shown to exert antimicrobial, antioxidant, anti-inflammatory, and anticancer activities. Its composition is influenced by seasonal, climatic and phytogeographic conditions. Further variability derives from the extraction methods. Multi Dynamic Extraction Method (MED) has been recently proposed to improve extracts reproducibility. Here, the cytotoxic/anticancer activity of three MED extracts of poplar-type propolis was assayed on human promyelocytic leukaemia HL60, human monocytic leukaemia THP-1, human osteosarcoma MG63, murine fibroblast L929 and human mesenchymal cells (hMSCs). As far as we are aware of, MG63 cells have never been challenged with propolis before, while few studies have so far addressed the effects of propolis on non-tumor cell lines. Consistent results were observed for all propolis preparations. The extracts turned out mildly cytotoxic toward cancer cells, in particular osteosarcoma cells (IC50: 81.9-86.7 µg/ml). Nonetheless, cytotoxicity was observed also in non-tumor L929 cells, with an even lower IC50. hMSCs demonstrated the lowest sensitivity to propolis (IC50: 258.3-287.2 µg/ml). In THP-1 cells, extracts were found to stimulate apoptosis caspase 3/7 activity. The IC50 values observed with osteosarcoma and leukaemia cells do not support a relevant cytotoxicity (as the figures abundantly exceeded 30 µg/ml), despites some selective activity exhibited with HL60 cells. The results confirm the validity of the extraction method, emphasizing the need to assess the selectivity of the interaction with cancer cells when screening for anticancer-drug candidates.

Description of a new group of variants of the Staphylococcus aureus elastin-binding protein that lacks an entire DNA segment of 180 bp.

The elastin-binding protein (EbpS) is a microbial surface component recognizing adhesive matrix molecule (MSCRAMM) found in Staphylococcus aureus that mediates bacterial cell binding to soluble elastin and tropoelastin. In scientific literature it is well established that the gene encoding for the elastin-binding protein (ebpS) is present in the vast majority of Staphylococcus aureus clinical isolates. The present study aimed at investigating a group of new variant forms of ebpS gene identified in S. aureus clinical strains isolated from implant-related orthopedic infections. A PCR screening for the ebpS gene, conducted on over two hundred S. aureus clinical isolates from implant-related infections revealed the detection of six strains exhibiting an altered amplicon size, shorter than expected. In order to elucidate the sequence changes present in these gene variants, the trait comprised between the primers was analyzed in all six isolates bearing the modification and in four isolates exhibiting the regular amplicon size. A similar form of the ebpS gene, lacking a DNA trait of 180 bp, was confirmed in all six isolates independently of their clonal origin. Interestingly, only three of these isolates, all with type IV polymorphism of the accessory genes regulator (agr) locus, showed exactly the same sequence and, thus, the same pattern of point mutations with respect to reference strains. From nucleotide translation, the corresponding encoded protein was found to lack an entire peptide segment of 60 amino acids. From nucleotide sequence translation, this modification was found to implicate the disappearance of an entire hydrophobic domain, whose functional significance needs to be further investigated.

Panton-Valentine leukocidin gene detected in Staphylococcus aureus strain isolated from a knee arthroprosthesis infection.

This report focuses on the molecular characterization of a Staphylococcus aureus strain isolated from a knee arthroprosthesis infection and recognized retrospectively as a carrier of the Panton-Valentine leukocidin gene. The stored microbiological isolate, which belonged to the strain collection of the Research Unit on Implant Infections of the Rizzoli Orthopaedic Institute, was retrieved for molecular analysis. Genotyping was carried out, revealing an interesting profile. In addition to the positivity for the Panton-Valentine toxin gene, the results indicated that the isolate belonged to the agr III group and was endowed with bbp and cna genes, both encoding for staphylococcal adhesins that bind bone proteins. The strain had the mecA gene for methicillin resistance, even though it was unable to resist any of the beta-lactam or other antibiotics. Its gene configuration matched that of other community-acquired methicillin-resistant and methicillin-susceptible Staphylococcus aureus(CA-MRSA and CA-MSSA) strains which have recently been reported worldwide. As far as we know,this is the first report on a PVL-positive S. aureus strain associated with an orthopedic implant (knee arthroprosthesis) infection.

Zirconium Carboxyaminophosphonate Nanosheets as Support for Ag Nanoparticles.

A layered insoluble inorganic-organic solid, namely zirconium phosphate glycine-N,N-bismethylphosphonate, was used to prepare dispersions of nanosheets to support active metals such as metallic silver nanoparticles and zinc ions. Zr phosphate-phosphonate microcrystals were first exfoliated with methylamine to produce a stable colloidal dispersion and then the methylamine was removed by treatment with hydrochloric acid. The obtained colloidal dispersion of Zr phosphate-phosphonate nanosheets was used to immobilize silver or zinc cations, via ion exchange, with the acidic protons of the sheets. The layered matrix showed a great affinity for the metal cations up taking all the added cations. The treatment of the dispersions containing silver ions with ethanol yielded metal silver nanoparticles grafted on the surface of the layered host. The samples were characterized by X-ray powder diffraction, elemental analysis transmission electron microscopy, and selected samples were submitted to antimicrobial tests. The nanocomposites based on Ag nanoparticles showed good bactericidal properties against the bacterial reference strain Staphylococcus epidermidis (S. epidermidis).

Antibacterial Properties of a Novel Zirconium Phosphate-Glycinediphosphonate Loaded with Either Zinc or Silver.

A novel compound consisting of a zirconium phosphate-glycinediphosphonate (ZPGly) has recently been introduced. This 2D-structured material forming nanosheets was exfoliated under appropriate conditions, producing colloidal aqueous dispersions (ZPGly-e) which were then loaded with zinc (Zn/ZPGly) or silver ions. Silver ions were subsequently reduced to produce metallic silver nanoparticles on exfoliated ZPGly nanosheets (Ag@ZPGly). In the search for new anti-infective materials, the present study investigated the properties of colloidal dispersions of ZPGly-e, Zn/ZPGly, and Ag@ZPGly. Ag@ZPGly was found to be a bactericidal material and was assayed to define its minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) on the five most prevalent pathogens of orthopaedic implant infections, namely: Staphylococcus aureus ATCC25923, Staphylococcus epidermidis RP62A, Enterococcus faecalis ATCC29212, Escherichia coli ATCC51739, and Pseudomonas aeruginosa ATCC27853. MIC and MBC were in the range of 125-250 µg/mL and 125-1000 µg/mL, respectively, with E. coli being the most sensitive species. Even colloidal suspensions of exfoliated ZPGly nanosheets and Zn/ZPGly exhibited some intrinsic antibacterial properties, but only at greater concentrations. Unexpectedly, Zn/ZPGly was less active than ZPGly-e.

Molecular epidemiology of Staphylococcus aureus from implant orthopaedic infections: ribotypes, agr polymorphism, leukocidal toxins and antibiotic resistance.

Staphylococcus aureus is a leading pathogen of implant-related infections. In the field of biomaterials a variety of alternative approaches are currently proposed for prophylaxis and treatment of implant infections, but little is known on the role of the different pathogenetic mechanisms and spreading strategies that lead selected S. aureus clones to prevail and become epidemic. This study aimed at identifying and characterizing the major clones in a collection of 200 S. aureus isolates from implant orthopaedic infections. Strain typing by automated ribotyping identified 98 distinct ribogroups. Ribogroups corresponded to specific accessory gene regulatory (agr) polymorphisms and possessed peculiar arrangements of toxins. The agr type II allele was more represented in epidemic clones, while agr type I in sporadic clones. A clear trend was observed, where epidemic clones resisted antibiotics more than sporadic ones. Conversely, the gene for lukD/lukE leukotoxin, found in 68% of the isolates, was unrelated to the level of clonal spreading. Surprisingly, the isolates of the most prevalent ribogroup were susceptible to almost all antibiotics and never possessed the lukD/lukE gene, thus suggesting the role of factors other than antibiotic resistance and the here investigated toxins in driving the major epidemic clone to the larger success.

New Parameters to Quantitatively Express the Invasiveness of Bacterial Strains from Implant-Related Orthopaedic Infections into Osteoblast Cells.

Complete eradication of bacterial infections is often a challenging task, especially in presence of prosthetic devices. Invasion of non-phagocytic host cells appears to be a critical mechanism of microbial persistence in host tissues. Hidden within host cells, bacteria elude host defences and antibiotic treatments that are intracellularly inactive. The intracellular invasiveness of bacteria is generally measured by conventional gentamicin protection assays. The efficiency of invasion, however, markedly differs across bacterial species and adjustments to the titre of the microbial inocula used in the assays are often needed to enumerate intracellular bacteria. Such changes affect the standardisation of the method and hamper a direct comparison of bacteria on a same scale. This study aims at investigating the precise relation between inoculum, in terms of multiplicity of infection (MOI), and internalised bacteria. The investigation included nine Staphylococcus aureus, seven Staphylococcus epidermidis, five Staphylococcus lugdunensis and two Enterococcus faecalis clinical strains, which are co-cultured with MG63 human osteoblasts. Unprecedented insights are offered on the relations existing between MOI, number of internalised bacteria and per cent of internalised bacteria. New parameters are identified that are of potential use for qualifying the efficiency of internalization and compare the behaviour of bacterial strains.

Antibiotic multiresistance strictly associated with IS256 and ica genes in Staphylococcus epidermidis strains from implant orthopedic infections.

In this study the presence both of the ica genes, encoding for biofilm exopolysaccharide production, and the insertion sequence IS256, a mobile element frequently associated to transposons, was investigated in relationship with the prevalence of antibiotic resistance among Staphylococcus epidermidis strains. The investigation was conducted on 70 clinical isolates derived from orthopedic implant infections. Among the clinical isolates investigated a dramatic high level of association was found between the presence of ica genes as well as of IS256 and multiple-resistance to all the antibiotics tested (oxacillin, penicillin, gentamicin, erythromycin, clindamycin, chloramphenicol, sulfamethoxazole + trimethoprim, ciprofloxacin, vancomycin). Noteworthy, a striking full association between the presence of IS256 and resistance to gentamicin was found, being none of the IS256-negative strain resistant to this antibiotic. This association is probably because of the link of the corresponding aminoglycoside-resistance genes, and IS256, often co-existing within the same staphylococcal transposon. In conclusion, in orthopedics, the presence of ica genes and that of IS256 in S. epidermidis genome should both be considered as informative markers of clinically relevant strains equipped with greatest and broadest resistance potential to survive to medical treatments.

Orthopedic implant infections: Incompetence of Staphylococcus epidermidis, Staphylococcus lugdunensis, and Enterococcus faecalis to invade osteoblasts.

Septic failure is still the major complication of prosthetic implants. Entering host cells, bacteria hide from host immune defenses, shelter from extracellular antibiotics, and cause chronic infection. Staphylococcus aureus, the leading etiologic agent of orthopedic implant infections, is able to enter bone cells and induce osteoblast apoptosis, osteoclast recruitment, and highly destructive osteomyelitis. Staphylococcus epidermidis, Staphylococcus lugdunensis, and Enterococcus faecalis are opportunistic pathogens causative of implant-related infections. This study investigated the ability to internalize into osteoblastic MG63 cells of 22 S. epidermidis, 9 S. lugdunensis, and 21 E. faecalis clinical isolates from orthopedic implant infections. Isolates were categorized in clusters by ribotyping. Internalization assay was carried out by means of a microtiter plate-based method. S. epidermidis, S. lugdunensis, and E. faecalis strains turned out incompetent to enter osteoblasts, exhibiting negligible internalization into MG63 cells, nearly three orders of magnitude lower than that of S. aureus. Osteoblast invasion does not appear as a pathogenetic mechanism utilized by S. epidermidis, S. lugdunensis, or E. faecalis for infecting orthopedic implants. Moreover, it can be inferred that intracellularly active antimicrobials should not be necessary against implant infections caused by the three bacterial species. Finally, implications with the uptake of biomaterial microparticles by nonphagocytic cells are enlightened. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 788-801, 2016.

Molecular Characterization of a Prevalent Ribocluster of Methicillin-Sensitive Staphylococcus aureus from Orthopedic Implant Infections. Correspondence with MLST CC30.

Staphylococcus aureus is the leading etiologic agent of orthopedic implant infections. Here a ribocluster of 27 S. aureus strains underwent further molecular characterization and subtyping by multilocus sequence typing (MLST) and spa-typing. This cluster had been detected by automated ribotyping (with the EcoRI restriction enzyme) of 200 S. aureus isolates from periprosthetic infections of patients who underwent revision at the Rizzoli Orthopaedic Institute. The ribocluster, consisting of agr type III strains, with a 74% co-occurrence of bone sialoprotein-binding (bbp) and collagen-binding (cna) genes, lacked mecA and IS256, and exhibited a high prevalence of the toxic shock syndrome toxin gene (tst, 85%). Strains' relatedness was analyzed by BURP and eBURST. Two predominant spa types, t012 (32%) and t021 (36%), and one predominant sequence type, ST30 (18/27, 67%) were identified: a S. aureus lineage spread worldwide belonging to MLST CC30. Two new sequence types (ST2954, ST2960) and one new spa type (t13129) were detected for the first time. Interestingly, the 27-strain cluster detected by ribotyping corresponded exactly to MLST CC30, the sole CC identified by eBURST.

Polysaccharide intercellular adhesin in biofilm: structural and regulatory aspects.

Staphylococcus aureus and Staphylococcus epidermidis are the leading etiologic agents of implant-related infections. Biofilm formation is the main pathogenetic mechanism leading to the chronicity and irreducibility of infections. The extracellular polymeric substances of staphylococcal biofilms are the polysaccharide intercellular adhesin (PIA), extracellular-DNA, proteins, and amyloid fibrils. PIA is a poly-ß(1-6)-N-acetylglucosamine (PNAG), partially deacetylated, positively charged, whose synthesis is mediated by the icaADBC locus. DNA sequences homologous to ica locus are present in many coagulase-negative staphylococcal species, among which S. lugdunensis, however, produces a biofilm prevalently consisting of proteins. The product of icaA is an N-acetylglucosaminyltransferase that synthetizes PIA oligomers from UDP-N-acetylglucosamine. The product of icaD gives optimal efficiency to IcaA. The product of icaC is involved in the externalization of the nascent polysaccharide. The product of icaB is an N-deacetylase responsible for the partial deacetylation of PIA. The expression of ica locus is affected by environmental conditions. In S. aureus and S. epidermidis ica-independent alternative mechanisms of biofilm production have been described. S. epidermidis and S. aureus undergo to a phase variation for the biofilm production that has been ascribed, in turn, to the transposition of an insertion sequence in the icaC gene or to the expansion/contraction of a tandem repeat naturally harbored within icaC. A role is played by the quorum sensing system, which negatively regulates biofilm formation, favoring the dispersal phase that disseminates bacteria to new infection sites. Interfering with the QS system is a much debated strategy to combat biofilm-related infections. In the search of vaccines against staphylococcal infections deacetylated PNAG retained on the surface of S. aureus favors opsonophagocytosis and is a potential candidate for immune-protection.

Staphylococcus lugdunensis, an aggressive coagulase-negative pathogen not to be underestimated.

The new emerging coagulase-negative pathogen Staphylococcus lugdunensis is responsible for severe cardiac and joint infections. Since the biochemical phenotypic systems designed for the identification of CoNS do not appear to be species specific and are hardly reliable for the discrimination of S. lugdunensis from other staphylococci, its precise identification requires fine molecular methods. The pathogenic mechanisms by which S. lugdunensis causes severe infections are not yet completely elucidated and in this review its virulence and toxic determinants are surveyed as well as its adhesins and biofilm production.