carla renata arciola

Abstract: The skin commensal and opportunistic pathogen Staphylococcus epidermidis is an important cause of nosocomial infections. Virulence is attributable to formation of biofilm, which provides a microenvironment that protects the bacterium from attack by the host immune system and by chemotherapy. In this study we extended to S. epidermidis strategies previously aimed at treatment of S. aureus biofilms using photodynamic treatment (PDT) combined with chemotherapy or phagocytosis. A significant reduction in bacterial survival was observed when structurally distinct biofilms were exposed to the cationic porphyrin, tetra-substituted N-methyl-pyridyl-porphine (TMP), and simultaneously to visible light. Of note, the extent of biofilm clearance depended on its maturation stage: developing, young biofilms, were more sensitive towards PDT than mature biofilms. Furthermore, PDT-treated biofilms exposed to vancomycin or subjected to phagocytic action of whole blood were almost completely eradicated. The data we obtained establish that PDT combined with antibiotics or host defenses may also be a useful approach for the inactivation of S. epidermidis biofilms.

Abstract: Molecular studies have cast light on new facts about the virulence factors of bacteria responsible for implant infections. Recently, the biofilm matrix has been shown to include a variety of important structural components, including DNA, in addition to polysaccharides and proteins. This finding has stimulated interest in substances able to disrupt biofilms by attacking both the poly-N-acetylglucosamine surface polysaccharide (PNAG) and the extracellular DNA (eDNA), namely, dispersin B, a PNAG-degrading enzyme, and DNase I. The therapeutic potential of these enzymes are reviewed in this issue of IJAO, as well as the ability of the excretions/secretions of the medicinal maggot Lucilia sericata to disrupt Staphylococcus epidermidis biofilms. The activity of bacterial proteases causes the release of eDNA, critical for the early development of biofilms. This complex process, including suicidal and fratricidal mechanisms, is also presented in the current issue of IJAO. The different sensitivities of S. aureus and S. epidermidis to enzymatic anti-biofilm agents and to the host's first line of defense, as well as the importance of knowing the cascade of regulatory genes in bacteria so as to interfere with biofilm production using gene therapy or quorum sensing inhibitors are also discussed in this issue. Other innovative approaches consist in disrupting biofilms by exposing them to photodynamic substances and simultaneously to visible light, and in coating biomaterial surfaces with organic molecules to prevent protein adsorption and biofilm formation.

Abstract: Staphylococcus aureus is a major, highly clonal, pathogen causing implant infections. This study aimed at investigating the diverse distribution of bacterial adhesins in most prevalent S. aureus strain types causing orthopaedic implant infections. 200 S. aureus isolates, categorized into ribogroups by automated ribotyping, i.e. rDNA restriction fragment length polymorphism analysis, were screened for the presence of a panel of adhesins genes. Within the collection of isolates, automated ribotyping detected 98 distinct ribogroups. For many ribogroups, characteristic tandem genes arrangements could be identified. In the predominant S. aureus cluster, enlisting 27 isolates, the bbp gene encoding bone sialoprotein-binding protein appeared a typical virulence trait, found in 93% of the isolates. Conversely, the bbp gene was identified in just 10% of the remaining isolates of the collection. In this cluster, co-presence of bbp with the cna gene encoding collagen adhesin was a pattern consistently observed. These findings indicate a crucial role of both these adhesins, able to bind the most abundant bone proteins, in the pathogenesis of orthopaedic implant infections, there where biomaterials interface bone tissues. This study suggests that specific adhesins may synergistically act in the onset of implant infections and that anti-adhesin strategies should be targeted to adhesins conjointly present.

Abstract: Staphylococcus epidermidis is an opportunistic pathogen of major clinical interest for its high prevalence in implant-associated infections. However, only little information is available on the phylogeny of its major clonal entities and their virulence and resistance markers. Therefore, purpose of this study was to characterize four clusters identified by automated ribotyping of 70 isolates derived from orthopedic, mostly foreign body-related infections. The ica locus, encoding the polysaccharide intercellular adhesin, the IS256 insertion element as well as the resistance to gentamicin, clindamycin, chloramphenicol, and ciprofloxacin were all traits uniquely observed in two of these clusters. The phylogenetic analysis of the S. epidermidis clusters offered a detailed insight into the clonal origin of exopolysaccharide- producing and multiresistant strains with transposons appearing to be actively involved in genetic exchanges.

Abstract: Staphylococcus aureus is a versatile and harmful human pathogen in both hospital- and community-acquired infections. S. aureus can initiate host infection by adhering to components of the extracellular matrix. Adherence is mediated by a variety of protein adhesins of the microbial surface component recognizing adhesive matrix molecule (MSCRAMM) family. In this article, we describe these MSCRAMMs in terms of structural organization and ligand-binding capacity and discuss their role as a possible target for immunotherapy.

Abstract: 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.

Abstract: Staphylococcus epidermidis, a main etiologic agent of implant-related infections, is showing increasing resistance to several antibiotic substances, among them the aminoglycosides, a class of drugs playing a relevant role in current medical protocols to prevent and treat clinical infections. Here we investigated the prevalence of aac(6')-Ie-aph(2''), aph(3')-IIIa, and ant(4') genes, encoding for the three forms of aminoglycoside-modifying enzymes (AME), responsible for resistance to aminoglycoside antibiotics, in 70 clinical isolates of S. epidermidis from orthopedic postsurgical and implant-related infections. In addition, ermA and ermC, the two most common staphylococcal genes conferring antibiotic resistance to macrolides, lincosamides, and streptogramin B (MLS(B)) were included in this investigation. All isolates were characterized by automated ribotyping, so that the presence of antibiotic resistance determinants was investigated in strains exhibiting different ribopatterns. Interestingly, combinations of coexisting AME genes appeared to be typical of specific ribopatterns. The aac(6')-Ie-aph(2'') gene was the most prevalent AME gene, being observed in 44% of the isolates. As far as the determinants for MLS(B) antibiotics are concerned, the ermC gene was observed in 33% of the isolates, while ermA was detected in a single isolate. These results provide a detailed characterization in terms of antibiotic resistance determinants of clones of S. epidermidis frequently isolated from implant orthopedic infections, providing useful indications for more effectual future strategies of infection prevention/eradication based on the incorporation of antibiotic drugs in biomaterials.

Abstract: DNA vaccines consist of a plasmid DNA genetically engineered to produce one or moreproteins able to elicit protective immune responses against virulence factors of infectious pathogens.Once introduced into the cells of the host, a DNA vaccine induces a high production of antigens bythe endogenous presence of the peptide codifying gene; improves antigen processing and presentation;may be able to simultaneously co-express multiple antigenic molecules; and, lastly, switches onboth humoral and cellular immune responses. In this mini-review, we underscore the advantageouscharacteristics of DNA vaccines compared with traditional ones and provide summaries of some of themore recent studies on them, mainly focusing the possibility of their use in targeting the staphylococcaladhesins that play a key role in the first adhesive phase of implant infections.

Abstract: Over the last few decades, the number of surgical procedures involving prosthetic materials has greatly multiplied, along with the rising medical and economic impact of implant-associated infections. The need to appropriately counteract and deal with this phenomenon has led to growing efforts to elucidate the etiology, pathogenesis and epidemiology of these types of infections, characterized by opportunistic pathogens. Molecular epidemiology studies have progressively emerged as a leading multitask tool to identify and fingerprint bacterial strains, unveil the complex clonal nature of important pathogens, detect outbreak events, track the origin of the infections, assess the clinical significance of individual strain types, survey their distribution, recognize associations of strain types with specific virulence determinants and/or pathological conditions, assess the role played by the specific components of the virulon, and reveal the phylogeny and the mechanisms through which new strain types have emerged. Despite the many advances that have been made thanks to these flourishing new approaches to molecular epidemiology, a number of critical aspects remain challenging. In this paper, we briefly discuss the current limitations and possible developments of molecular epidemiology methods in the investigation and surveillance of implant infections.

Abstract: PURPOSE: Identification of putative new virulence factors as additional targets for therapeutic approaches alternative to antibiotic treatment of multi-resistant enterococcal infections. METHODS: The EF3314 gene, coding for a putative surface-exposed antigen, was identified by the analysis of the Enterococcus faecalis V583 genome for LPXTG-motif cell wall anchor surface protein genes. A non-polar EF3314 gene deletion mutant in the E. faecalis 12030 human clinical isolate was obtained. The wild type and the isogenic mutant strain were investigated for biofilm formation, adherence to Hela cells, survival in human macrophages and a Caenorhabditis elegans infection model. The aminoterminal portion of the EF3314 protein was overexpressed in E. coli to obtain mouse polyclonal antibodies for use in Western blotting and immunolocalization experiments. RESULTS: The EF3314 gene has an unusually high GC content (46.88% vs. an average of 37.5% in the E. faecalis chromosome) and encodes a protein of 1744 amino acids that presents a series of 14 imperfect repeats of 90 amino acids covering almost the entire length of the protein. Its global organization is similar to the alpha-like protein family of group B streptococci, enterococcal surface protein Esp and biofilm associated protein Bap from S. aureus. The EF3314 gene was always present and specific for E. faecalis strains of human, food and animal origin. Differences in size depended on variable numbers of repeats in the repetitive region. CONCLUSIONS: EF3314 is a newly described, surface exposed protein that contributes to the virulence properties of E. faecalis.

Abstract: Adherence of oral bacteria to the surface of dental restorative materials is considered an important step in the development of secondary caries and periodontal disease. The aim of this study was to investigate and compare the adherence of different restorative materials to Streptococcus mutans strain (CCUG35176) in order to ascertain possible differences. The materials tested ranged across different classes including: flowable composites (Gradia Direct LoFlo; Filtek Supreme XT Flowable), anterior composites (Gradia Direct Anterior), universal composites (Filtek Supreme XT), packable composites (Filtek Silorane; Filtek P60), glass-ionomers (Fuji IX Gp Extra; Equia) and a control reference material (Thermanox plastic coverlips). Bacterial suspension was deposited onto each material and the adhesion was evaluated trough the colony forming units (CFUs) determination. Packable silorane-based composite was found to be less adhesive than posterior packable composite P60, flowable composites and glass ionomers. The fluoride of glass ionomers did not prevent the attachment of S. mutans; furthermore, after roughness analysis and SEM investigations, the hypothesis that the difference in bacterial adhesion can be determined by the particular surface chemistry of the material itself as well as by different electrostatic forces between bacteria and restorative surfaces must be given serious consideration.

Abstract: Infection still represents one of the most serious and ravaging complications associated with prosthetic devices. Staphylococci and enterococci, the bacteria most frequently responsible for orthopedic postsurgical and implant-related infections, express clinically relevant antibiotic resistance. The emergence of antibiotic-resistant bacteria and the slow progress in identifying new classes of antimicrobial agents have encouraged research into novel therapeutic strategies. The adoption of antisense or "antigene" molecules able to silence or knock-out bacterial genes responsible for their virulence is one possible innovative approach. Peptide nucleic acids (PNAs) are potential drug candidates for gene therapy in infections, by silencing a basic gene of bacterial growth or by tackling the antibiotic resistance or virulence factors of a pathogen. An efficacious contrast to bacterial genes should be set up in the first stages of infection in order to prevent colonization of periprosthesis tissues. Genes encoding bacterial factors for adhesion and colonization (biofilm and/or adhesins) would be the best candidates for gene therapy. But after initial enthusiasm for direct antisense knock-out or silencing of essential or virulence bacterial genes, difficulties have emerged; consequently, new approaches are now being attempted. One of these, interference with the regulating system of virulence factors, such as agr, appears particularly promising.

Abstract: This report focuses on the molecular characterization of a Staphylococcus aureus strainisolated 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 collectionof the Research Unit on Implant Infections of the Rizzoli Orthopaedic Institute, was retrievedfor molecular analysis. Genotyping was carried out, revealing an interesting profile. In addition tothe positivity for the Panton-Valentine toxin gene, the results indicated that the isolate belonged tothe agr III group and was endowed with bbp and cna genes, both encoding for staphylococcal adhesinsthat bind bone proteins. The strain had the mecA gene for methicillin resistance, even thoughit was unable to resist any of the beta-lactam or other antibiotics. Its gene configuration matched thatof 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 (kneearthroprosthesis) infection.

Abstract: Enterococcus faecalis is an opportunistic pathogen, which today represents one of the leading aetiologic agents of nosocomial infections and, increasingly, of implant infections. Here, in a collection of 43 E. faecalis isolated from implant orthopaedic infections, virulence-related phenotypes (biofilm and gelatinase production) and genotypes (gelE and esp) were studied to characterize epidemic clones identified and grouped by ribotyping. The presence of the esp gene and a marked and steady biofilm formation ability appeared to be the features associated with the clonal spreading, as well as a conspicuous gelatinase production, whereas the simple presence of gelE appeared non-specific of the epidemic clones. Antibiotic multi-resistance and strong biofilm production abilities together with a high phenotypic expression of gelatinase are an important equipment of E. faecalis to colonize peri-prosthesis tissues and to spread out as causative agents of implant orthopaedic infections.

Abstract: 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.

Abstract: The role of biofilm in implant infections and the genetic control of its production are still giving rise to great interest and controversial new results are continuing to emerge from widespread investigations. Bacteria embedded in biofilms are more resistant to most antibiotics. Hypotheses regarding the mechanisms of antibiotic multi-resistance in biofilm are brought to light, including the onset of persister cells during the attachment of bacteria to implant surfaces.This 2008 special issue ''Focus on Implant Infections'', presents new strategies for combating biofilm infections,such as the use of staphylococcal quorum-sensing inhibitors or the ''bioelectric effect'': the phenomenon by which electric current can enhance the activity of some antimicrobial agents against bacteria in biofilms.

Abstract: Research on implant infections requires cooperative efforts and integration between basic and clinical expertises. An international group of women scientists is acting together in this field. The main research topics of the participants of this group are described. Formation of bacterial biofilms, antibiotic resistance and production of virulence factors like adhesins and toxins are investigated. New biomaterials, coatings and drugs designed to inhibit microbial adhesion are evaluated, and infection-resistant biomaterials are under study, such as a novel heparinizable polycarbonate-urethane (Bionate) or incorporation of diamino-diamide-diol (PIME) to reduce bacterial attachment. The correlation between biofilm production and the accessory-gene-regulator (agr) is investigated in Staphylococcus aureus. The ability to form biofilm has also been shown to be one of the important virulence factors of Enterococcus faecalis, favouring colonization of inert and biological surfaces. The study of quorum sensing has led to the discovery of a quorum sensing inhibitor termed RIP that suppresses staphylococcal biofilm and infections. The immune response and the local defence mechanisms of the host against implant-associated infections, activation and infiltration of immunocompetent cells into the sites of infection have been studied in patients with implant-associated osteomyelitis. Production of monoclonal antibodies (mAbs) as possible vaccines against the staphylococcal collagen-binding MSCRAMMs is in progress.

Abstract: Implant-related infections are broadly recognized as one of the most serious and devastating complications associated with the use of biomaterials in medical practice. The growing interest and need for the development of implant materials with reduced susceptibility to microbial colonization and biofilm formation has necessitated the development of a series of in vitro and in vivo models for evaluation and preclinical testing. Current technologies provide these investigations with an ample choice of qualitative and quantitative techniques for an accurate assessment of the bioactivity and anti-infective efficacy of any new compound or device. These tests are typically performed using a reference bacterial strain designated as the test or reference strain. Recent molecular epidemiological studies have identified the complex clonal nature of most prevalent etiological agents implicated in implant-associated infections. New information which is continually emerging on the identity and the characteristics of both sporadic and epidemic clones must be considered when selecting a reference. A new emerging requirement is that the strain should be representative of the clones causing clinically relevant infections; they should, therefore, belong to the most prevalent epidemic clones rather than to sporadic ones, which may occur in only 1 out of 200 infections or even fewer. The correct choice of reference strain for preclinical tests is of crucial importance for the clinical significance of the achieved results. In this paper we report our experience and recommendations regarding this issue.

Abstract: Photodynamic treatment (PDT) has been proposed as a new approach for inactivation of biofilms associated with medical devices that are resistant to chemical additives or biocides. In this study, we evaluated the antimicrobial activity of merocyanine 540 (MC 540), a photosensitizing dye that is used for purging malignant cells from autologous bone marrow grafts, against Staphylococcus epidermidis biofilms. Effect of the combined photodynamic action of MC 540 and 532 nm laser was investigated on the viability and structure of biofilms of two Staphylococcus epidermidis strains, RP62A and 1457. Significant inactivation of cells was observed when biofilms were exposed to MC 540 and laser simultaneously. The effect was found to be light dose-dependent but S. epidermidis 1457 biofilm proved to be slightly more susceptible than S. epidermidis RP62A biofilm. Furthermore, significant killing of both types of cells was attained even when a fixed light dose was delivered to the biofilms. Confocal laser scanning microscope (CLSM) analysis indicated damage to bacterial cell membranes in photodynamically treated biofilms, while disruption of PDT-treated biofilm was confirmed by scanning electron microscopy (SEM).

Abstract: Orthopedic implants may fail owing to different reasons: poor osseointegration at the tissue-implant interface, generation of wear debris, stress and strain imbalance between implant and surrounding tissues, and infections. To ensure success in orthopedics, implant materials must not evoke an undesirable inflammatory response, they must be habitable by bone-forming cells (favoring adhesion of osteoblasts), hinder formation of soft connective tissue (hindering adhesion of fibroblasts), and be anti-infective (discouraging bacterial adhesion). Recent studies have suggested that nanophase materials have a better efficacy as bone implants in favoring osseointegration compared to conventional orthopedic implant materials. This minireview discusses studies on nanophase materials as bone implants, focusing on the effect of these materials in inhibiting bacterial adhesion for the prevention of implant infections.

Abstract: The Alpha-like protein (Alp) family, repeat-containing surface proteins once thought to be important adhesion factors confined to pathogenic streptococci and enterococci, is broader than previously known. Analysis of the annotated microbial genomes has identified new potential members of the Alp family not only in other Gram- positive opportunistic pathogens but also in commensal microflora of the human gut and the skin. This finding has highlighted the importance of genome sequencing projects for unraveling in greater detail lateral gene transfer events involving virulence factors between pathogens and commensals. These should receive constant attention not only as part of infectious disease prevention programs, but also in the food and biotechnology industries.

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Abstract: Implant infection remains the major and often irreducible complication in clinical use of biomaterials, demanding new therapeutic and preventive strategies. Etio-pathogenesis of biomaterials-related infections is being more and more studied, and various virulence bacterial factors have progressively been identified, but little is still known about the weight of the distinct molecules in the context of specific peri-implant infection sites. Molecular epidemiology has become recently integrated into the research on implant infections. What distinguishes molecular epidemiology from the simple molecular biology is that the use of molecular techniques is applied to the study of the distribution and prevalence of virulence and resistance genes in collections of bacterial clinical isolates from implant infections. Here, the authors comment on the range of molecular techniques available, reviewing the various applications of molecular epidemiology to the study of implant infections and providing some experimental examples related to the field of orthopaedic implant infections. They highlight the new opportunities arising from molecular epidemiology of designing measures useful to prevent and treat implant infections. The knowledge of the relative weight of virulence factors and of their regulatory mechanisms at molecular level can open the way to new strategies also including gene therapies aimed at silencing or knocking out crucial genes responsible for the aggressive tools (adhesins, biofilm production, antibiotic resistance) of the aetiological agents of implant-related infections.

Abstract: Enterococcus faecalis is an emerging etiologic agent of hospital infections, exhibiting high rates of antibiotic resistances. Here, 43 isolates of E. faecalis, taken from patients with implant orthopaedic infections come at the Rizzoli Orthopaedic Institute from 13 different Italian regions, were genotyped by an automated RiboPrinter and analyzed for antimicrobial susceptibility. The three most represented ribogroups were the iris-ribogroup, with its nine strains, the daisy-ribogroup, containing eight isolates, and the violet-ribogroup, with five isolates. The isolates belonging to the iris-ribogroup interestingly share a basal antibiotic resistance pattern, all being resistant to tetracycline, gentamicin and erythromycin. Among the isolates belonging to the daisy-ribogroup, 3 out of 8 were multi-resistant, 2 of which with the same pattern. More varied appeared the resistance profiles of the violet-ribogroup, in which 2 out of the five isolates were multi-resistant, the other being only bi- or mono-resistant. Noteworthy was also the variety of geographic origins and of implant infection sites for all the isolates. Cluster analysis demonstrated that ribogroups had a high internal similarity and that the three largest ones belonged to well-defined clusters, highlighting the tendency of E. faecalis to give rise to resistant clones in orthopaedic peri-implant infections.

Abstract: The status of population density is communicated among bacteria by specific secreted molecules, called pheromones or autoinducers, and the control mechanism is called ""quorum-sensing"". Quorum-sensing systems regulate the expression of a panel of genes, allowing bacteria to adapt to modified environmental conditions at a high density of population. The two known different quorum systems are described as the LuxR-LuxI system in gram-negative bacteria, which uses an N-acyl-homoserine lactone (AHL) as signal, and the agr system in gram-positive bacteria, which uses a peptide-tiolactone as signal and the RNAIII as effector molecules. Both in gram-negative and in gram-positive bacteria, quorum-sensing systems regulate the expression of adhesion mechanisms (biofilm and adhesins) and virulence factors (toxins and exoenzymes) depending on population cell density. In gram-negative Pseudomonas aeruginosa, analogs of signaling molecules such as furanone analogs, are effective in attenuating bacterial virulence and controlling bacterial infections. In grampositive Staphylococcus aureus, the quorum-sensing RNAIII-inhibiting peptide (RIP), tested in vitro and in animal infection models, has been proved to inhibit virulence and prevent infections. Attenuation of bacterial virulence by quorum-sensing inhibitors, rather than by bactericidal or bacteriostatic drugs, is a highly attractive concept because these antibacterial agents are less likely to induce the development of bacterial resistance.

Abstract: 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.

Abstract: Staphylococci have become the most common cause of nosocomial infections, especially in patients with predisposing factors such as indwelling or implanted foreign polymer bodies. The pathogenesis of foreign-body associated infections with S.aureus and S. epidermidis is mainly related to the ability of these bacteria to form thick, adherent multilayered biofilms. In a biofilm, staphylococci are protected against antibiotic treatment and attack from the immune system, thus making eradication of the infections problematic. This necessitates the discovery of novel prophylactic and therapeutic strategies to treat these infections. In this review, we provide an overview of staphylococcal biofilm components and discuss new possible approaches to controlling these persistent biofilm-dwelling bacteria.

Abstract: Antibiotic treatment of infections associated with the use of indwelling medical devices in ageing and/or severely ill patients represents a significant healthcare problem due to the difficulty of treating such infections and to the various collateral effects that may be observed following the often aggressive therapy. We summarize some effects of antibiotics on the expression of virulence factors of the microorganisms which cause such infections. These effects, particularly those resulting in a stimulation of bacterial virulence, might be usefully included among the other well-known collateral effects of antibiotic therapy.

Abstract: In recent years, a variety of new technologies have been proposed that allow rapid qualitative and quantitative microbiological analyses. In this paper we discuss the urgent needs for reliable and rapid microbiological analytical techniques in different applicative fields involving the research, production and medical application of implant materials, and the potential benefits derived from the use of new methods for rapid bacterial quantification. Current compendial methods are easy to perform and have gained confidence over their long period of use, but the supplemental use of new technologies could represent real breakthroughs whenever sensitive and rapid responses are urgently required and not met by the tests currently in use. Overall, the new microbiological methods require critical evaluation depending on their specific type of application and they may still not be thought of as totally substitutive, but they certainly exhibit considerable potential for different areas of biomaterials, as well as for advanced therapy medicinal and tissue engineering treatments.

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Abstract: Staphylococcus aureus has emerged as a major cause of implant infections. It is known that it is able to produce several toxins that contribute to its armory of virulent weapons, but there are still no data on their prevalence among isolates recovered from biomaterial-centered infections. In this study, 200 Staphylococcus aureus isolates from infections related to different types of orthopedic implants (hip and knee arthroprostheses, internal and external fixation devices) were tested by polymerase chain reaction for the prevalence of genes encoding for leukotoxins. Although almost all isolates were positive for the ã-hemolysin gene (99%), none was positive for lukM. The leukotoxin genes lukE/lukD were found in 67% of isolates. The presence of lukE/lukD was significantly associated with that of Accessory Gene Regulatory locus agr II. The lukE/lukD-positive isolates were significantly more prevalent in the staphylococcal isolates from knee arthroprostheses than in the isolates from the other implant types. The genes encoding Panton-Valentine leukocidin components were detected in only one isolate that, curiously enough, was taken solely from a knee arthroprosthesis infection.

Abstract: Microorganisms universally attach to surfaces, resulting in biofilm formation. These biofilms entail a serious problem in daily clinical practice because of the great prevalence of implantable device-related infections. Differences in antibiotic activity against planktonic and sessile bacteria may relate to clinical failures in the treatment of biofilm-related infections (BRI). Bacteriophages have several characteristics that make them potentially attractive therapeutic agents in some selected clinical settings, like for example BRI. They are highly specific and very effective in lysing targeted bacteria, moreover, they appear to be safe for humans. Many studies have shown the potential of phages for the treatment of infectious diseases in plants and animals, including infections with highly drug-resistant bacteria. The therapeutic use of bacteriophages, possibly in combination with antibiotics, may be a valuable approach in BRI. However, many important questions still remain that must be addressed before phages can be endorsed for therapeutic use in humans.

Abstract: The vast use of prosthetic materials in medicine over the last decades has been accompanied by the appearance of new opportunistic pathogens previously considered incapable of causing infections with significant morbidity and/or mortality. In this regard, the genus Staphylococcus enlisting numerous species usually characterized by a saprophytic habit covers a special role. Apart from Staphylococcus aureus and Staphylococcus epidermidis, well known for their large prevalence in implant-related infections, a number of further staphylococcal species are progressively being indicated for their pathogenic potential. The increasing attention on these opportunistic bacteria is due to an ever growing number of clinical reports, which is also deriving from a more accurate identification of these species with currently available techniques. This synopsis intends to offer an overview on recently emerging coagulase-negative staphylococci (CoNS) as well as coagulase-positive/-variable staphylococci exhibiting distinct traits of virulence, pathogenicity, and epidemiologic impact depending among others on the medical field, the type of prosthetic device and its anatomic location.

Abstract: Several species belonging to Staphylococcus genus (non Sau/ non Sep species) exhibit increasing abilities as opportunistic pathogens in colonisation of periprosthesis tissues. Here we report on antibiotic resistance of 193 strains, belonging to non Sau/ non Sep species, consecutively collected from orthopedic implant infections in a period of about 40 months. The 193 strains (representing 17% of all staphylococci isolated) were analysed for their antibiotic resistance to 16 different drugs. Five species turned out more prevalent, ranging from 1 to 5%: S. hominis (4.2%), S. haemolyticus (3.7%), S. capitis (2.7%), S. warneri (2.6%), and S. cohnii (1.6%). Among these, the prevalence of antibiotic resistance to penicillins was similar, ranging from 51% to 66%. Conversely, significant differences were observed for all the remaining antibiotics. For S. haemolyticus the resistances to oxacillin and imipenem, the four aminoglycosides and erythromycin were at least twice that of the other three species which were compared. S. warneri was on the contrary the species with the lowest occurrence of resistant strains. Ten species appeared only rarely at the infection sites: S. lugdunensis, S. caprae, S. equorum, S. intermedius, S. xylosus, S. simulans, S. saprophyticus, S. pasteuri, S. sciuri, and S. schleiferi. The behaviours of these species, often resistant to penicillins, were individually analysed. Differences in both the frequencies and the panels of antibiotic resistances observed among the non Sau/ non Sep species: i) suggest that horizontal spreading of resistance factors, if acting, was not sufficient per se to level their bio-diversities; ii) highlight and confirm the worrisome appearance within the Staphylococcus genus of emerging ""new pathogens"", not homogeneous for their virulence and antibiotic resistance prevalence, which deserve to be recognised and treated individually.

Abstract: Enterococci are opportunistic pathogens which today represent one of the leading causes of nosocomial infections. We have examined a collection of 52 Enterococcus faecalis isolated from orthopedic infections to determine if they were characterized by a specific pattern of virulence factors. The isolates were evaluated for biofilm formation, presence of genes coding the enterococcal surface protein (esp) and gelatinase (gelE), as well as for gelatinase production. While the rate of esp-positive isolates was comparable to that found among strains from other clinical sources, we found a significantly higher rate of strong biofilm formers and gelatinase producers. Particularly high was the rate of gelE-carrying strains expressing the gene. Data suggest that these two factors in particular may play an important role in enterococcal infections associated with biomaterials.

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Abstract: Over the last 15 years, with the advent of modern standards in the control of sterility within the operating room environment and adequate protocols of peri-operative antibiotic prophylaxis, the incidence of infections associated to orthopedic implants has become very low. Nevertheless, the event of infection still represents one of the most serious and devastating complications which may involve prosthetic devices. It leads to complex revision procedures and, often, to the failure of the implant and the need for its complete removal. In orthopedics, for the enormous number of surgical procedures involving invasive implant materials, even if nowadays rare, infections have a huge impact in terms of morbidity, mortality, and medical costs. The difficult battle to prevent and fight bacterial infections associated to prosthetic materials must be played on different grounds. A winning strategy requires a clear view of the pathogenesis and the epidemiology of implant-related infections, with a special attention on the alarming phenomenon of antibiotic resistance. In this regard staphylococci are the prevalent and most important causative pathogens involved in orthopedic implant-related infections, and, thus, the main enemy to defeat. In this paper, we offer an overview of the complexity of this battleground and of the current and new, in our opinion most promising, strategies in the field of biomaterials to reduce the risks and counteract the establishment of implant infections.

Abstract: There are numerous reports in the literature using animal models of osteomyelitis for investigating pathogenesis, diagnosis, and treatment of bone infections. Rabbits, rats, and dogs are commonly used animals, and, less frequently, chickens, guinea pigs, miniature pigs, goats, and sheep. Commonly used bones for creating local osteomyelitis include tibia, femur, and radius, and, less frequently, mandible and spine. When designing a specific model, one should consider which animal and which bone will be used, which route for inoculation (either local injection or systemically through vascular injection), which bacterial species and how many bacteria should be applied, if and what sclerosing agent, foreign body or implant should be employed, and if local trauma is needed. Basic methods of evaluation include clinical observation, radiography, microbiology, and histology.

Abstract: One-hundred and twenty-eight enterococcal isolates were examined for their ability to form biofilm in relation to the presence of the gene encoding the enterococcal surface protein (esp), production of gelatinase and to the source of isolation. Neither esp nor gelatinase seemed to be required for biofilm formation: both Enterococcus faecalis and Enterococcus faecium did not show a correlation between the presence of either esp or the production of gelatinase and biofilm formation. However, in E. faecium while esp was found in isolates from either source, the presence of both esp and biofilm together was only found in strains from clinical settings, suggesting that there exists a synergy between these factors which serves as an advantage for the process of infection.

Abstract: Stainless steel is a metallic alloy largely employed in orthopaedics. However, the presence in its composition of a high quantity of nickel, an agent known to trigger toxic and allergic responses, is cause for concern. In this study, we have investigated the in vitro biocompatibility of a new nickel-reduced stainless steel, namely Böhler P558, in comparison to the conventional stainless steel AISI 316L. The neutral red (NR) uptake and the amido black (AB) tests were performed on L929 fibroblasts and MG63 osteoblasts to assess the cytotoxicity, while cytogenetic effects were evaluated on CHOK1 cells by studying the frequency of Sister Chromatid Exchanges (SCE) and chromosomal aberrations. Ames test was used to detect the mutagenic activity. The expression of selected markers typical of differentiated osteoblasts, such as alkaline phosphatase activity (ALP), type I collagen (CICP) and osteocalcin (OC) production, were also monitored in MG63 cells cultured on the tested materials. Our results indicate the absence of significant cytotoxicity and genotoxicity for both test alloys. ALP, CICP and OC analyses confirmed that both materials support the expression of these phenotypic markers. Overall, these data show that this Ni-free alloy possesses good in vitro biocompatibility and could have a potential for orthopaedic applications.

Abstract: Biofilm-forming ability is increasingly being recognized as an important virulence factor in Staphylococcus epidermidis. This study compares three different techniques for the detection of biofilm-positive strains. The presence of icaA and icaD genes responsible for biofilm synthesis was investigated by a PCR method in a collection of 80 S. epidermidis strains isolated from orthopedic implant infections. The results from molecular analysis were compared with those obtained by two classic phenotypic methods, the Congo red agar (CRA) plate test and the microtiter plate test (MtP). Fifty-seven percent of all the examined strains were found icaA/icaD-positive, of which only three were not positive for CRA test. Differently, by the MtP method, 66% of the strains were found to be biofilm-producers but only a limited agreement with the PCR-method was noticeable because of the observation of (icaA/icaD+)/MtP- strains (8%) and of a surprising ambiguous result of (icaA/icaD-)/MtP+ strains (16%). The category of the weak biofilm-producers provided the highest contribution to these mismatching results (10%). The better agreement between the CRA plate test with the molecular detection of ica genes indicates the former as a reliable test for the phenotypic characterization of virulence of clinical isolates. However, MtP method remains a precious tool for the in vitro screening of different biomaterials for the adhesive properties using a reference strain.

Abstract: Streptococcus agalactiae is an etiological agent of several infective diseases in humans. We previously demonstrated that FbsA, a fibrinogen-binding protein expressed by this bacterium, elicits a fibrinogen-dependent aggregation of platelets. In the present communication, we show that the binding of FbsA to fibrinogen is specific and saturable, and that the FbsA-binding site resides in the D region of fibrinogen. In accordance with the repetitive nature of the protein, we found that FbsA contains multiple binding sites for fibrinogen. By using several biophysical methods, we provide evidence that the addition of FbsA induces extensive fibrinogen aggregation and has noticeable effects on thrombin-catalyzed fibrin clot formation. Fibrinogen aggregation was also found to depend on FbsA concentration and on the number of FbsA repeat units. Scanning electron microscopy evidentiated that, while fibrin clot is made of a fine fibrillar network, FbsA-induced Fbg aggregates consist of thicker fibers organized in a cage-like structure. The structural difference of the two structures was further indicated by the diverse immunological reactivity and capability to bind tissue-type plasminogen activator or plasminogen. The mechanisms of FbsA-induced fibrinogen aggregation and fibrin polymerization followed distinct pathways since Fbg assembly was not inhibited by GPRP, a specific inhibitor of fibrin polymerization. This finding was supported by the different sensitivity of the aggregates to the disruptive effects of urea and guanidine hydrochloride. We suggest that FbsA and fibrinogen play complementary roles in contributing to thrombogenesis associated with S. agalactiae infection.

Abstract: Several species belonging to Staphylococcus genus, other than Staphylococcus aureus and Staphylococcus epidermidis (non Sau/ non Sep species), exhibit increasing abilities as opportunistic pathogens in the colonisation of periprosthetic tissues. Consequently, the availability of means for accurate identification is crucial to assess the pathogenic characteristics and to clarify clinical relevance of the individual species. Here, 146 clinical staphylococcal isolates belonging to non Sau/ non Sep species from prosthesis-associated orthopedic infections were analyzed by conventional enzymatic galleries and by automated ribotyping. Twelve different species were recognised: S. capitis, S. caprae, S. cohnii, S. equorum, S. haemolyticus, S. hominis, S. lugdunensis, S. pasteuri, S. sciuri, S. simulans, S. warneri, S. xylosus. Ribotype identifications were compared with the phenotypes obtained by the Api 20 Staph system and/or ID 32 Staph system. ID 32 Staph profiles were more consistent with ribotyping results than Api Staph profiles. Across the different staphylococcal species investigated, correct identifications with Api Staph were 45%, while with ID 32 Staph they were 59%. It has, however, to be mentioned that ID 32 Staph was mostly applied to discriminate unmatched ribotyping and Api Staph identifications, thus to a subpopulation of strains with ""atypical"" metabolic profile. Automated ribotyping provided a correct identification for 91% of the isolates. These results confirm automated ribotyping as a convenient rapid technique, still subject to improvements, which will accurately and rapidly recognise the newly emerging staphylococcal pathogens in implant-related orthopedic infections.

Abstract: BACKGROUND: In a previous study, a number of genes, associated with spine musculoskeletal deformity phenotypes in mouse and in synteny between mouse and man, were identified as candidate genes for IS. Among these genes, MATN1, which carries a polymorphic microsatellite marker within its sequence, was selected for a linkage analysis. MATN1 is localised at 1p35 and is mainly expressed in cartilage. The objective of this study was to assess a linkage disequilibrium between the matrilin-1 (MATN1) gene and the idiopathic scoliosis (IS). METHODS: The genetic study was conducted on a population of 81 trios, each consistent of a daughter/son affected by idiopathic scoliosis (IS) and both parents. In all trios components, the region of MATN1 gene containing the microsatellite marker was amplified by a polymerase chain reaction. The amplicons were analysed by a DNA sequencer-genotyper. The statistical linkage analysis was performed using the extended transmission/disequilibrium test. RESULTS: Three microsatellite polymorphisms, respectively consisting of 103 bp, 101 bp and 99 bp, were identified. ETDT evidenced a significant preferential transmission for the 103 bp allele (Chi-square = 5.058, df = 1, P = 0.024) CONCLUSION: The results suggest that the familial idiopathic scoliosis is associated to the MATN1 gene.

Abstract: The production and the mechanism of action of exotoxins from Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa are presented. The attack to the immune host's defenses is the main virulence factor of opportunistic bacteria in implant infections, favoring the invasion and colonization of compromised periprosthesis tissues.

Abstract: In recent years the progress in the field of nanotechnologies has offered new possibilities to control the superficial features of implant materials down to a nanoscale level. Several studies have therefore tried to explore the effects of nanostructured biomaterial surfaces on the behavior of eukaryotic cells. However, nanotopography could exert an influence also on the behavior of prokaryotic cells, with relevant implications concerning the susceptibility of implant surfaces to infection. Aim of this study was to examine the behavior of Staphylococcus aureus on polyethylene terephthalate (PET) surfaces either cylindrically nanostructured (PET-N) or flat ion-etched (PET-F), and on tissue culture-grade polystyrene (PS). Microbial adherence was assessed by chemiluminometry under 4 different conditions: (a) bacteria suspended in MEM medium, (b) bacteria in MEM supplemented with 10% fetal bovine serum (FBS), (c) test surfaces preconditioned in FBS, and (d) post-exposure of colonised surfaces to serum-supplemented MEM. Under all circumstances, PET-F and PET-N specimens showed identical bacterial adhesion properties. In the absence of serum, all 3 test materials showed a very high adhesivity to microbial cells and both PET surfaces exhibited greater adhesion than PS. On the contrary, the presence of 10% serum in solution significantly affected cell behavior: the number of microbial cells on all surfaces was drastically reduced, and the adhesion properties of PET surfaces with respect to PS were reversed, with PET being less adhesive. Overall, the specific cylindrical nanostructures created on PET did not significantly influence microbial behavior. Ongoing studies are verifying whether other nanotopographies with different geometry could have more substantial effects.

Abstract: Titanium-based implants are successfully used for various biomedical applications. However, in some cases, e.g. in dental implants, failures due to bacterial colonization are reported. Surface modification is a commonly proposed strategy to prevent infections. In this work, titanium oxide, naturally occurring on the surface of titanium, was modified by promoting the formation of a mixed titanium and zinc oxide, on the basis of the idea that zinc oxide on titanium surface may act as the zinc oxide used in pharmaceutical formulation for its lenitive and antibacterial effects. The present work shows that it is possible to form a mixed titanium and zinc oxide on titanium surfaces, as shown by Scanning Electron Microscopy and XPS analysis. To this end titanium was preactivated by UV on crystalline titanium oxide, both in the anatase form or in the co-presence of anatase and rutile. By performing antibacterial assays, we provide evidence of a significant reduction in the viability of five streptococcal oral strains on titanium oxide surfaces modified with zinc. In conclusion, this type of chemical modification of titanium oxide surfaces with zinc might be considered a new way to reduce the risk of bacterial colonization, increasing the lifetime of dental system applications.

Abstract: In natural and man-made environments, microbial communities thrive as biofilms on living (e.g. tissue) and inanimate (e.g. plastic, metal, wood, mineral) surfaces. Biofilms are found in a wide range of aqueous habitats, including physiological fluids. Numerous types of microorganisms are able to colonize catheters, implants, prosthetics, and other medical devices manufactured from different metallic and non-metallic materials dwelling within a human body. The development of biofilm is facilitated by the production of extracellular polymeric substances (EPS). Biofilms formed on surfaces of metallic materials may alter interfacial electrochemical processes, which can lead to increased corrosion of the colonized substratum. Deterioration of metallic materials in the presence of a biofilm is termed biocorrosion or microbially-influenced corrosion (MIC). In the field of biomaterials, ""biocorrosion"" is commonly used when describing the effect of host tissue on the corrosion of implant metals and alloys. Therefore, to avoid confusion, we will here use the term MIC as a reference to biofilm-influenced corrosion. It is important to realise that although most metals are prone to microbial colonization, i.e. to biofouling, this does not imply that they are susceptible to MIC. For example, a metal such as titanium, accumulates biofilm, however, it still demonstrates excellent resistance against MIC. Corrosion is, by definition, an electrochemical process, therefore, electrochemical techniques are frequently employed to determine and measure the rate of abiotic, as well as biologically driven corrosion reactions. This communication addresses the use of electrochemical techniques for monitoring (i) biofilm formation on and (ii) MIC of implant metals and alloys.

Abstract: The possibility of biosurfaces with high or low adhesiveness for protein, bacteria or eukaryotic cells is discussed. At the interface surface object/biological milieu, biocompatibility, (no) bioadhesion and (no) biocontamination are shown to be correlated with physico-chemical surface characteristics. First consequence is the (no) possibility of biofilm formation. Substrates with low surface energy could interact only with hydrophobic biomolecules. On substrates with high surface energy, a water monolayer spontaneously formed. Modification of a surface by plasma techniques is a way for engineering biomaterials. Plasma techniques are dry processes and more suitable for biomedical applications. In the field of biomaterial medical devices, in hygienic prevention of nosocomial diseases, in food packaging, the use of substrates with a very hydrophilic character may help to prevent the proliferation of cells and bacteria. Such a technique is so efficient that antibiotic molecules are not necessary. Therefore, surface engineering is a tool for modifying and adapting materials to specific biological applications.

Abstract: The opportunistic pathogen Staphylococcus epidermidis is able to produce biofilm and to frequently cause implant infections. In recent years, it has also exhibited an increasing antimicrobial drug resistance. Here, the resistance to a panel of 16 different antibiotics in 342 clinical strains of S. epidermidis from orthopaedic implant infections has been investigated. The isolates were pheno- and genotyped for extracellular polysaccharide production, relevant to staphylococcal biofilm formation, in order to ascertain possible associations with antibiotic resistance. Approximately 10% of the isolates were found to be sensitive to all screened antibiotics. In all, 37-38% were resistant to beta-lactams such as oxacillin and imipenem, while the resistance to penicillin, ampicillin, cefazolin, cefamandole, was consistently observed in over 80% of the strains. Erythromycin- and clindamycin- resistant strains were approximately 41% and 16%, respectively. Of the isolates, 10% was resistant to chloramphenicol, 23% to sulfamethoxazole and 26% to ciprofloxacin. Resistance to vancomycin was never observed. Interestingly, exopolysaccharide-producing strains exhibited a significantly higher prevalence in the resistance to the four aminoglycosides (gentamicin, amikacin, netilmicin, tobramycin), to sulfamethoxazole and to ciprofloxacin with respect to non-producing isolates. Moreover, multiple resistance to antibiotics was more frequent among exopolysaccharide-forming strains.

Abstract: Stainless steel is a metallic alloy largely employed in orthopedics, maxillofacial surgery and orthodontic therapy. However, the presence in its composition of a high quantity of nickel, an agent known to trigger toxic, allergic and cancerogenous responses in humans, is cause of some concern. In this study, we have investigated the in vitro mutagenicity and genotoxicity of a new nickel-free stainless steel, namely P558, in comparison to the conventional stainless steel AISI 316L. The cytogenetic effects were evaluated by studying the frequency of Sister Chromatid Exchanges (SCE) and chromosomal aberrations. Ames test was performed to detect the mutagenic activity. Both P558 and AISI 316L did not cause any significant increase in the average number of SCE and in chromosomal aberrations, either with or without metabolic activation. Furthermore, the Ames test showed that the extracts of both P558 and of AISI 316L are not mutagenic. Overall, these findings prove that P558 is devoid of genotoxicity and mutagenicity. The present results, together with other previous interesting observations that P558 promotes osseointegration, suggest that this new nickel-free stainless steel can represent a better alternative to other conventional steel alloys.

Abstract:

Abstract: Here are reported data on virulence determinants of Staphylococcus aureus from orthopedic surgical infections, emphasizing on the genes encoding fibronectin (fnbA, fnbB) and collagen (cna) adhesins. 191 S. aureus strains from orthopedic infections (53 from internal fixation devices, 29 external fixation devices, 15 knee arthroprostheses, 30 hip arthroprostheses, 45 surgical reconstruction and 19 non-associated to medical devices) were investigated for the presence of the genes of the collagen-binding protein Cna and of the two fibronectin-binding proteins, FnbA and FnbB. 87 (46%) strains were found to be cna+ without significant variations across the different surgical categories considered. Conversely, the fnbA and the fnbB genes were almost always present in all surgical categories. The finding that, among the investigated adhesins, fibronectin-adhesins are present in the majority of the implant associated S. aureus clinical isolates encourages the development of strategies to specifically block the interaction of bacteria with matrix fibronectin by antagonist ligands.

Abstract: In Staphylococcus epidermidis, ica locus encodes for the synthesis of a polysaccharide intercellular adhesin (slime or biofilm). A multiplex polymerase chain reaction (PCR) for the detection of the five individual genes of ica locus was developed, with the aim to probe the set of genes in a large collection of Staphylococcus epidermidis clinical isolates. Single representative fragments for icaR, icaA, icaD, icaB, and icaC genes were selected. Multiplex PCR was applied to two reference Staphylococcus epidermidis strains [the non-biofilm-forming ATCC 12228 and the biofilm-forming ATCC 35984 (RP62A)] and to 400 clinical isolates of Staphylococcus epidermidis from orthopedic prosthesis associated infections. The gene profile was compared with the phenotypic biofilm-forming ability, evaluated by means of an optimized Congo red agar (CRA) plate test. Among the clinical isolates, 228 (57%) turned out completely ica positive and were biofilm producing. Among the 172 non-biofilm-forming strains (43%), 164 (41%) were completely ica negative and 8 strains (2%) harbored all five ica genes. The ica locus thus proves to be a cluster of strictly linked genes, without any evidence of single gene deletion.

Abstract: Implant infection is an aggressive, often irreducible post-surgical infection. It remains the primary cause of implant failure. Bacterial contamination during surgery and subsequent adhesion onto biomaterial surface of opportunistic microorganisms, such as staphylococcal species, exopolysaccharidic slimes or specific adhesins, initiates the implant infection. Pathogenesis of periprosthestic infection is the focus of studies aimed at developing infection resistant materials.

Abstract: This article concisely reviews the effects of sterilization on the mechanical properties and surface chemistries of implantable biomaterials. This article also summarizes the biological effects of the sterilization-related changes in the implant. Because there are so many different types of implant materials currently in use (including metals, polymers, and diverse biological materials), the response of tissue to these different materials varies dramatically. This review further discusses the effects of sterilization on in vivo and in vitro tissue response specifically to implantable metals and polyethylene, with the possibility of future biocompatibility testing of the implants sterilized with supercritical phase carbon dioxide sterilization.

Abstract: A significant proportion of medical implants become the focus of a device-related infection, difficult to eradicate because bacteria that cause these infections live in well-developed biofilms. Biofilm is a microbial derived sessile community characterized by cells that are irreversibly attached to a substratum or interface to each other, embedded in a matrix of extracellular polymeric substances that they have produced. Bacterial adherence and biofilm production proceed in two steps: first, an attachment to a surface and, second, a cell-to-cell adhesion, with pluristratification of bacteria onto the artificial surface. The first step requires the mediation of bacterial surface proteins, the cardinal of which is similar to S. aureus autolysin and is denominated AtlE. In staphylococci the matrix of extracellular polymeric substances of biofilm is a polymer of beta-1,6-linked N-acetylglucosamine (PIA), whose synthesis is mediated by the ica operon. Biofilm formation is partially controlled by quorum sensing, an interbacterial communication mechanism dependent on population density. The principal implants that can be compromised by biofilm associated infections are: central venous catheters, heart valves, ventricular assist devices, coronary stents, neurosurgical ventricular shunts, implantable neurological stimulators, arthro-prostheses, fracture-fixation devices, inflatable penile implants, breast implants, cochlear implants, intraocular lenses, dental implants. Biofilms play an important role in the spread of antibiotic resistance. Within the high dense bacterial population, efficient horizontal transfer of resistance and virulence genes takes place. In the future, treatments that inhibit the transcription of biofilm controlling genes might be a successful strategy in inhibiting these infections.A significant proportion of medical implants become the focus of a device-related infection, difficult to eradicate because bacteria that cause these infections live in well-developed biofilms. Biofilm is a microbial derived sessile community characterized by cells that are irreversibly attached to a substratum or interface to each other, embedded in a matrix of extracellular polymeric substances that they have produced. Bacterial adherence and biofilm production proceed in two steps: first, an attachment to a surface and, second, a cell-to-cell adhesion, with pluristratification of bacteria onto the artificial surface. The first step requires the mediation of bacterial surface proteins, the cardinal of which is similar to S. aureus autolysin and is denominated AtlE. In staphylococci the matrix of extracellular polymeric substances of biofilm is a polymer of beta-1,6-linked N-acetylglucosamine (PIA), whose synthesis is mediated by the ica operon. Biofilm formation is partially controlled by quorum sensing, an interbacterial communication mechanism dependent on population density. The principal implants that can be compromised by biofilm associated infections are: central venous catheters, heart valves, ventricular assist devices, coronary stents, neurosurgical ventricular shunts, implantable neurological stimulators, arthro-prostheses, fracture-fixation devices, inflatable penile implants, breast implants, cochlear implants, intra-ocular lenses, dental implants. Biofilms play an important role in the spread of antibiotic resistance. Within the high dense bacterial population, efficient horizontal transfer of resistance and virulence genes takes place. In the future, treatments that inhibit the transcription of biofilm controlling genes might be a successful strategy in inhibiting these infections.

Abstract: Enterococci are commensals of human and animal intestinal tract that have emerged in the last decades as a major cause of nosocomial infections of bloodstream, urinary tract and in infected surgical sites. Enterococcus faecalis is responsible for ca. 80% of all enterococcal infections while Enterococcus faecium accounts for most of the others; among the most relevant risk factors for development of enterococcal infections is the presence of implanted devices. The pathogenesis of such infections is poorly understood, but several virulence factors have been proposed. Among them, the ability to form biofilm has recently been shown to be one of the most prominent features of this microorganism, allowing colonization of inert and biological surfaces, while protecting against antimicrobial substances, and mediating adhesion and invasion of host cells and survival within professional phagocytes. Biofilm formation has been shown to be particularly important in the development of prosthetic valve enterococcal endocarditis and stent occlusion. Enterococci are also able to express other surface factors that may support colonization of both inert and biological surfaces, and that may be involved in the invasion of, and survival within, the host cell.

Abstract: In the last decade the rising phenomenon of resistance to most common antibiotic drugs among staphylococcal clinical isolates has been a reason for serious concern and alarm. The present study investigated the prevalence of antibiotic resistance within a large microbial collection including 530 clinical strains of S. aureus and 408 strains of S. epidermidis to a panel of 16 different drugs. All strains were isolated from orthopedic infections, either associated or non-associated with implant materials. Interestingly, our data show that the profile of the prevalence of antibiotic resistance within the two species of pathogens is extremely similar for the vast majority of the drugs screened. The only statistically significant variations in prevalence concerned, in order of relevance, the following 5 out of 16 antibiotics: sulfamethoxazole (in combination with trimethoprim), erythromycin, and, to a lesser extent, oxacillin, imipenem, and clindamycin. In the case of Staphylococcus aureus, the isolates associated to implant materials were found more frequently resistant to all 4 aminoglycosides screened as well as to ciprofloxacin.

Abstract: In spite of the recent achievements derived from modern protocols of prophylaxis, orthopedic surgical infections still remain unacceptably frequent, especially in light of the often devastating outcomes of septic complications. The spectrum and the prevalence of the bacteria most frequently involved in orthopedic infections are here explored, with particular reference to those infections associated to implant biomaterials, which were grouped based on device typology. During a 30 months period (from September 2000 to April 2003), 1027 microbial strains were consecutively isolated from 699 patients undergoing revision surgery at the Rizzoli Orthopedic Institute. 775 (75.5%) of all these microorganisms were identified as belonging to the Staphylococcus genus, 82 (8%) to the Enterobacteriaceae family, 75 (7.3%) to the Pseudomonas genus, 54 (5.3%) to the Enterococcus genus and 20 (1.9%) to the Streptococcus genus. While confirming the importance of staphylococci as the most diffuse cause of infection, our data indicate an unexpectedly high prevalence of S. epidermidis on infected hip and knee arthroprostheses, respectively of 42% and 44%. The spectrum of bacteria infecting either internal or external fracture fixation devices appears to differ from that of hip and knee arthroprostheses and more closely resembles that of infections non-associated to medical devices, being characterized by a relatively higher prevalence of Staphylococcus aureus (over 40%) and Pseudomonas aeruginosa. Enterobacteriaceae and members of the Streptococcus and Corynebacterium genera are frequently associated with implants in which surgical incisions were made near the perineum, determining a completely altered spectrum.

Abstract: Bacterial adhesion to the surface of composite resins and other dental restorative materials is an important parameter in the aetiology of secondary caries formation. The aim of the present study was to investigate the adhesion of a Streptococcus mutans strain (ATCC 25175) on the surface of different restorative materials. The test materials examined included three flowable composites (Filtek Flow, Tetric Flow, and Arabesk Flow), three microhybrid composites (Clearfil APX, Solitaire 2, and Z250), two glass-ionomers (Fuji IX, Fuji IX fast), a compomer (F2000), an ormocer (Admira), and a control reference material (tissue culture grade, surface treated polystyrene). The adhesion tests were carried out in 24-well plates. Quantitative turbidimetric measurements were finally performed in order to indirectly evaluate the amount of bacteria retained on the material surface after in vitro exposure to the bacteria suspension. Under these conditions, with the exception of the Admira ormocer and the Fuji IX fast glass ionomer, which were found to be more adhesive, all the other material surfaces showed a similar susceptibility to bacterial adhesion, exhibiting values not significantly different than the reference polystyrene control. Furthermore, the release of fluoride from some of the test surfaces did not appear capable to reduce early bacterial adhesion.

Abstract: Staphylococcus epidermidis biofilm-forming strains produce a polysaccharide intercellular adhesin (PIA), which mediates bacterial cell aggregation and favours the colonisation on prosthetic implants. PIA synthesis is regulated by the icaADBC locus. In vitro, by repeated subcultures of a biofilm-producing strain, the loss of the ability to produce biofilm appears associated with the insertion of the IS256 element into the ica locus. This study was aimed (i) to investigate if the five genes of ica locus are always all present in different strains of S. epidermidis, and (ii) to search if IS256 insertion naturally occurs in ica locus without making recourse to the experimental procedure of repeated subcultures of strains. 120 S. epidermidis clinical isolates from peri-prosthesis infections were investigated both by an original multiplex PCR analysis of the ica genes and by PCR amplification of the IS256 element. Also two reference strains (the biofilm-negative S. epidermidis ATCC 12228 and the biofilm-forming ATCC 35984 [RP62A]) and two biofilm-negative RP62A-derived acriflavin mutants (D9 and HAM892) were analysed. D9 e HAM892 were for the first time shown to contain in ica locus, at the base 3319, a 1300-bp insertion with a DNA sequence corresponding to IS256. Among the 120 clinical isolates, 51 (43%) turned out completely ica-positive, 69 completely ica-negative (57%). The genes of the ica locus appear, in all cases of the present collection, strictly linked each other, so they are either all present or all absent. In this collection, IS256 was present in eight out of the 69 ica-negative strains and in 34 out of the 51 ica-positive strains. IS256, also when present in bacterial genomic DNA, was never found inside the ica locus, thus suggesting that insertion/excision of this element is not a natural occurring mechanism for off/on switching of biofilm production.

Abstract: Attention has recently been paid to identify and elucidate those pathogenetic mechanisms, which play a significant role in sustaining the early phases of Staphylococcus epidermidis colonisation and infection development. Several analogies with the physiology of Staphylococcus aureus, a more thoroughly investigated pathogen, have lead to carefully consider all bacterial surface components that mediate cell adhesion. This study aimed at investigating the presence of the fbe gene encoding for a fibrinogen-binding protein in a collection of 107 S. epidermidis strains isolated from orthopaedic infections and 67 from central venous catheter-associated infections. The strains isolated from orthopaedic infections were in large part associated to four different classes of orthopaedic devices, respectively: internal fixation devices, external fixation devices, knee arthroprostheses and hip arthroprostheses. The molecular epidemiology analysis performed by PCR enlightened a statistically significant difference in the prevalence of this adhesion mechanism between orthopaedic infections and catheter-related infections, respectively, of 78% and 91%. The prevalence of fbe ranged from 67% to 91%, suggesting that, even though this adhesin is not strictly necessary for the development of infection, nevertheless it represents a rather common characteristic of strains causing clinical infections, this independently on the presence or the absence of implant materials.

Abstract: BACKGROUND & OBJECTIVES: Enterococci are important nosocomial pathogens that are increasingly difficult to treat due to intrinsic and acquired resistance to antibiotics. Studies were taken up to identify virulence factors and to characterise pathogenic mechanisms of such infections to evaluate potential targets for treatments alternative to antibiotic therapy. This study was carried out to evaluate the contribution of extracellular polysaccharide expressed by Enterococcus faecalis to resistance to phagocytosis and survival within rat peritoneal macrophages. METHODS: Six E. faecalis clinical isolates were tested for their ability to survive within rat peritoneal macrophages. Cytochalasin D, colchicine and monodansylcadaverine were used to investigate the route of enterococcal entry inside macrophages. RESULTS: Four of the isolates were able to produce extracellular polysaccharide and form biofilm after growth in glucose-supplemented medium, while no production could be detected in glucose deficient medium. Two isolates were polysaccharide-negative in both conditions. Isolates expressing extracellular polysaccharide were able to survive for more than 24 h compared to polysaccharide-negative bacterial cells of the same strain grown in glucose-deficient medium, which were readily cleared. Cytochalasin D virtually abolished the number of viable intracellular bacteria, after growth in either trypticase soy broth (TSB) or TSB supplemented with glucose; colchicine and monodansylcadaverine strongly affected survival of polysaccharide-positive bacteria, significantly more than that of polysaccharide-negative ones. INTERPRETATION & CONCLUSION: Biofilm-forming E. faecalis survived within rat peritoneal macrophages significantly better than polysaccharide-negative isolates. Perturbators of cytoskeleton and of surface receptors turnover, indicated receptors-mediated endocytosis as the most likely route for enterococcal entry into macrophages.

Abstract: Eleven isolates of Enterococcus faecalis causing endocarditis were screened for possible virulence factors with PCR and phenotypic assays. The gene coding for the enterococcal surface protein (esp) was detected in one isolate only, and haemolysin was produced by two isolates. Aggregation substance, biofilm formation and gelatinase were present in seven, nine and eight isolates, respectively. Predisposing factors, particularly hospitalisation and multiple antibiotic therapy, appeared to be more relevant to the development of enterococcal endocarditis following bloodstream infections than the pattern of virulence factors.

Abstract: Among ceramic materials, recent interest has been risen on fluorinated hydroxyapatites, which undergo slow in vivo degradation and offer a more stable interface for osseointegration and bone fixation. Apart from the information available on the chemistry, little is known of their biological properties and, more specifically, of their interaction with bone cells. The aim of the present study has been to investigate the behaviour and differentiation of human bone marrow-derived mesenchymal cells cultured on two substrata consisting of fluorohydroxyapatite (FHA)-coated titanium and characterised by different surface roughness. To this end, osteoprogenitor cells were seeded on the test materials and, once adhered, were induced to differentiate to osteoblastic cells. The cell behaviour on the different surfaces was monitored for a period of up to 2 weeks. The results obtained indicate that FHAs are cytocompatible materials, which allow the adhesion and growth of osteogenic cells. Mesenchymal cells promptly adhered, covering the surface of the test materials, and subsequently expressed some typical markers of osteoblasts. No significant difference in alkaline phosphatase specific activity was observed when comparing the two test material surfaces to plastic control. At 7 days the number of adhered cells and the presence in the medium of C-terminal propeptide of type I collagen appeared lower or slightly lower for cultures on FHA substrata than on plastic control. These differences tended to subside with time.

Abstract: Staphylococcus epidermidis is able to adhere onto biomaterials and to cause implant infections. Recently, host matrix proteins, which in vivo cover the implants, have been indicated as substrates for adhesion by specific bacterial adhesins. Here, the binding of S. epidermidis to fibronectin, a main protein of the extracellular matrix, and the effect of heparin on this interaction were studied by dynamic force spectroscopy (DFS). Novelties are that S. epidermidis strains analysed by DFS were clinical isolates from prosthesis-associated infections, genotyped and phenotyped for their adhesion properties to fibronectin and examined as living cells. Thus, fibronectin-binding staphylococci adhered to the fibronectin-coated substratum and formed a continuous layer assuring their contact with the fibronectin-coated cantilever tip during the approach-retraction cycles of the DFS measurements. Results show that only a single molecular binding site of fibronectin is involved in the interaction with S. epidermidis, that it takes place at the domain near the C-terminus and that it is specifically inhibited by heparin.

Abstract: Fibronectin (Fn) is an important mediator of bacterial invasions and of persistent infections like that of Staphylococcus epidermis. Similar to many other types of cell-protein adhesion, the binding between Fn and S. epidermidis takes place under physiological shear rates. We investigated the dynamics of the interaction between individual living S. epidermidis cells and single Fn molecules under mechanical force by using the scanning force microscope. The mechanical strength of this interaction and the binding site in the Fn molecule were determined. The energy landscape of the binding/unbinding process was mapped, and the force spectrum and the association and dissociation rate constants of the binding pair were measured. The interaction between S. epidermidis cells and Fn molecules is compared with those of two other protein/ligand pairs known to mediate different dynamic states of adhesion of cells under a hydrodynamic flow: the firm adhesion mediated by biotin/avidin interactions, and the rolling adhesion, mediated by L-selectin/P-selectin glycoprotein ligand-1 interactions. The inner barrier in the energy landscape of the Fn case characterizes a high-energy binding mode that can sustain larger deformations and for significantly longer times than the correspondent high-strength L-selectin/P-selectin glycoprotein ligand-1 binding mode. The association kinetics of the former interaction is much slower to settle than the latter. On this basis, the observations made at the macroscopic scale by other authors of a strong lability of the bacterial adhesions mediated by Fn under high turbulent flow are rationalized at the molecular level.

Abstract: Staphylococcus epidermidis is a frequent pathogen in infections associated with orthopedic implants. We studied 123 S. epidermidis strains from infections related to orthopedic implants, as regards their ability to express a factor of virulence, namely the slime, an extracellular polysaccharide, which mediates adherence to implants and bacterial colonization. The slime-producing ability was determined by PCR detection of icaA and icaD genes responsible for slime synthesis, and by culture on Congo red agar plates in which slime-producing strains form black colonies, while nonslime-forming ones develop red colonies. 56% of the S. epidermidis isolates were icaA- icaD-positive and grew to become black colonies. In the evaluation of the distribution of slime-forming strains in different sites and types of implants, we found a slight, but not statistically significant, increase in slime-forming strains in total joint prostheses, where tissue compression near the articular faces can form niches in which bacteria crowd, sheltered by the slime. Our findings confirm the role of ica genes as a virulence marker in the pathogenesis of implant-associated orthopedic infections. However, they do not show the existence of a higher frequency of slime-positive strains in a specific type of implant.

Abstract: The chances of integration between an implant and the surrounding bone tissue depend on the surface characteristics of the implant itself. Particularly, chemical composition and surface roughness of the material have emerged as crucial factors in affecting the behaviour of cells in contact with the material. Among various surfaces, calcium phosphate coatings seem to favour a rapid initial integration, but their dissolution by extracellular fluids raises some concern about the long-term stability at the bone-implant interface. Fluorinated apatites are known to be more stable than other ceramic coatings, but, at present, little is known on their effects on human cells. In this study, MG63 osteoblast-like cells were seeded onto two fluorohydroxyapatite (FHA)-coated titanium alloy (Ti6Al4V) materials differing in roughness, respectively, LR-FHA (Ra = 5.6 microm) and HR-FHA (Ra = 21.2 microm). Quantification of the cells in contact with the FHA-coated materials by conventional methods involved some technical difficulties, on which we report. Only the indirect esteem by the measure of total content of proteins and a procedure based on cell count, following a double enzymatic treatment to detach the cells, offered plain results, indicating no significant differences between cellular growth in contact with test materials and with plastic control. Differentiation and functionality of the cells were comparatively evaluated by analysis of alkaline phosphatase activity and osteocalcin production. As far as osteocalcin release is concerned, only slight variations were detected on FHA-coated materials in comparison with the control. Both types of coatings showed a significant increase in alkaline phosphatase activity with respect to the control, the roughest surface exhibiting a more prolonged effect on the time.

Abstract: The G-->T mutation at base 1 of intron 1 at the binding site of the Sp1 transcription factor of the collagen type I alpha 1 gene (COLIA1, GenBank accession no. AF017178) is a putative marker for low bone mineral density and osteoporotic fractures. A new method for the detection of this mutation is presented, based on the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), which utilizes two separate and simultaneous PCRs to detect the normal and mutated alleles. The forward primer (positions 1307-1336 of the gene) is common to both amplifications. Two reverse primers (positions 1566-1546) are used, differing in the 3' base (3'-C for the normal S allele and 3'-A for the mutated s allele). The former amplification uses the reverse primer specific for the S allele; the latter uses the reverse primer with the 3'-base complementary to the mutated base of the s allele. In the SS condition, amplification occurs only in the former reaction and in the ss condition only in the latter. Both reactions give a product in the Ss condition. Direct DNA sequencing of a COLIA1 region containing the G-->T polymorphism demonstrates the validity of this ARMS-PCR method. The new method is more reliable than a previously published detection method, which utilizes a mismatched reverse primer, introducing a restriction site in the T-substituted (s) allele. However, the restriction enzyme is costly, its digestion time long, and incomplete digestion can lead to an underestimation of the frequency of ss homozygosity. The latter can result in incorrect conclusions about a linkage between osteoporosis and the COLIA1 polymorphism. In a survey of the COLIA1 polymorphism in 133 osteoporotic subjects with femur fractures, 7 cases of ss homozygosity were consistently detected both by direct DNA sequencing and by the ARMS-PCR method. This, in contradistinction to the mismatched-primer method by which 3 of these 7 cases were inaccurately diagnosed as Ss heterozygosities.

Abstract: A number of studies appears to give emphasis to the role of prosthetic materials in determining microbial adherence and resistance to host defence and drug therapy. Aim of this study was to explore whether the direct contact with biomaterial substrata of different chemical nature could influence bacterial behaviour, determining possible changes in the bacteria population as far as antibiotic resistance is concerned. To this end, susceptibility to penicillin, erythromycin, clindamycin, cefamandole, imipenem, vancomycin, ciprofloxacin. ampicillin, cefazolin, trimethoprim-sulfamethoxazole, chloramphenicol, amikacin and netilmicin was evaluated in a methicillin-, gentamicin- and tobramycin-resistant Staphylococcus epidermidis strain, after in vitro adhesion to polymethylmethacrylate (PM MA) and to silicone elastomer. The susceptibility to antibiotics of both adherent bacteria and bacteria which, although exposed to the materials, had not undergone adhesion was measured as bacterial growth inhibition area onto a plate antibiogram. according to Kirby-Bauer and using an image analyser system. The results obtained suggest that the two test materials considered in this study were capable to condition bacterial behaviour. In particular. the adhesion onto PMMA surfaces induced a marked and significant decrease in susceptibility to the following beta-lactam antibiotics: cefamandole (32%), cefazolin (23%), imipenem (27%), ampicillin (31%). Moreover, PMMA caused a lower but significant reduction in resistance to vancomycin (15%), chloramphenicol (16%), amikacin (13%). netilmicin (13%), erythromycin (11%) trimethoprim-sulfamethoxazole (13%). In contrast, the adhesion onto silicone elastomer appeared to influence bacterial changes to a lesser extent and elicited a significant decrease in susceptibility only to cefazolin (10%) and amikacin (11). Further studies are required to thoroughly investigate the mechanisms of these variations, even though, also according to other authors, one of the best conceivable conclusions is that some material substrata can lead to selection of variant adhesive bacteria with increased antibiotic resistance.

Abstract: Recently, it has been shown that S. epidermidis includes the ica operon responsible for slime production. In the operon, coexpression of icaA and icaD genes is required for full slime synthesis. In this study, the presence of icaA and icaD genes was searched for in a collection of 100 Staphylococcus epidermidis strains from catheter-associated infections by an original PCR method. Another 51 strains of S. epidermidis isolated from the skin or mucosa of healthy volunteers (26 of which derived from the hospital staff) were also investigated. Slime-forming ability was phenotypically tested on Congo red agar plates. Sixty-one percent of the strains isolated from catheters were icaA- icaD-positive and produced slime. The results indicate that detection of ica genes by a PCR method is a useful tool for prompt identification of S. epidermidis slime-forming strains isolated from catheter-related infections. Also, three saprophytic strains from the hospital staff were positive for slime synthesis and presence of ica genes, suggesting a potential diffusion of slime-forming strains in hospital personnel.

Abstract: This investigation was conduced on a collection of 113 S. epidermidis strains isolated from biomaterial-associated infections. All strains were examined both for the presence of icaA and icaD genes responsible for slime synthesis by a PCR method and for the in vitro slime production ability by the Congo red agar (CRA) plate test. In the present study, the original CRA test was optimised adopting a six-colour reference scale for a fine classification of colonies colours. The six-colour tones of the scale were as follows: very black (vb), black (b), almost black (ab), which were considered as positive results, and bordeaux (brd), red (r), and very red (vr), interpreted as negative. 57.5% of all the strains were found to be icaA icaD-positive as well as slime-forming onto CRA, exhibiting the following colonies colours: vb (35.4%); b (15.9%); ab (6.2%). The percentage of icaA icaD-negative strains was 42.5% and all of them were negative onto CRA: brd (19.5%), r (14.2%), vr (8.8%). The comparison of colour classification with the information on ica genes confirmed the validity of the scale adopted, providing support to the criteria used for a correct interpretation of the colonies colour during the execution of the CRA test. Overall these results indicate a fine consistency between these two experimental methods and a good reliability of CRA plate test, especially when this is supported by a colourimetric scale.

Abstract: The formation of biofilm represents an important virulence factor of certain strains of Staphylococcus epidermidis and S. aureus. The ability of bacteria to aggregate, forming biofilms, is strictly related to the capacity of producing an extracellular mucoid substance often referred to as slime, whose main component is of polysaccharidic nature and consists of glycosaminoglycans. In recent years, new molecular techniques based on PCR have come alongside more traditional methods for identification of virulent biofilm-forming strains. The detection of the genes governing the production of such extracellular polysaccharide and, in particular, the icaA, the icaC and the icaD genes, provides us with a rapid and accurate technique for strain characterization. However, well-established methods, such as the Congo red agar test are still needed in order to confirm the phenotypic expression in the case of possible phase-variant strains. In future, the complete knowledge of the genetic mechanisms of phenotype modulation, comprehending all regulatory genes, could permit the characterization of the isolates just by molecular means in a single step.

Abstract: The allele frequency of the G-->T mutation of COL1A1 gene (collagen type I alpha 1 gene, GenBank accession n. AF017178) was analyzed by a newARMS-PCR method in 240 osteoporotic subjects bearing a femoral neck fracture. The method is based on the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). Normal and mutated alleles were detected by two PCRs, in which a common forward primer (positions 1307 to 1336 of the gene) and two reverse primers (positions 1566 to 1546), differing in the 3'-base (3'-C for the normal Sallele and 3'-A for the mutated sallele) are used. In the SS condition, amplification occurs only in one of the two PCRs, and in the ss condition only in the other. In the Ss condition both reactions give a product. This ARMS-PCR method avoids the use of any restriction enzyme, as described by Grant and colleagues in a previously published method based on a mismatched reverse primer which introduced a restriction site in the T-substituted (s) allele and in a method recently proposed by Vinkanharju and co-workers. In a survey for COL1A1 polymorphism in 240 osteoporotic subjects with femur fractures, here presented, a frequency of 80.6% was found for the G allele and 19.4% for the T allele. There were 66.7% dominant SS subjects, 27.9% Ss heterozygotes and 5.4% ss recessive homozygotes.

Abstract: The antibiotic-resistance both of adherent bacteria to polymethylmethacrylate (PMMA) and of bacteria which, although exposed to the material, had not undergone adhesion, was measured as bacterial growth inhibition area onto a plate antibiogram, according to Kirby-Bauer and using a dedicated image analyzer system. The adhesion onto PMMA induces a marked (about 30%) increase in resistance to beta-lactam antibiotics (cefamandole, cefazolin, imipenem and ampicillin) and a lower (about 15%) but significant increase to the macrolide erythromycin, to two aminoglycosides (amikacin, netilmicin) and to vancomycin, chloramphenicol and trimethoprim-sulfamethoxazole.

Abstract: Bacterial adhesion to polymethylmethacrylate and to silicon elastomer, materials frequently used in clinical applications, has been investigated to assess whether adhesion selects methicillin-resistant mutants in the bacterial population in contact with the materials. The methicillin susceptibility of a susceptible Staphylococcus aureus (ATCC 25923) was measured by a modification of plate antibiogram Kirby-Bauer method, which allows optimised detection of small variations in antibiotic susceptibility. In both adherent and non-adherent bacterial subpopulations, the presence of mecA gene, which encodes for the protein PBP 2a responsible for methicillin resistance was searched for by Polymerase Chain Reaction (PCR). The contact with the two polymers did not induce in the bacteria population any phenotypic increase in methicillin resistance, or the selection of mutants carrying the mecA gene.

Abstract: In periprosthesis tissues, Staphylococcus epidermidis produces extracellular polysaccharide slime. Recently it has been shown that S. aureus also produces slime and that both S. epidermidis and S. aureus contain the ica operon responsible for slime production. In the operon, icaA encodes for N-acetylglutaminyltransferase, the enzyme for polysaccharide synthesis. However, co-expression of icaA and icaD is required for full slime synthesis. The slime-producing strains of both S. epidermidis and S. aureus are more virulent and are responsible for severe postsurgical or periprosthesis infections. The authors describe a simple, rapid, and reliable polymerase chain reaction method to detect icaA and icaD. The method was applied to the detection of ica genes on two reference strains, 15 strains each of S. epidermidis and S. aureus from periprosthesis infections and 10 strains from the skin and mucosa of healthy volunteers. icaA and icaD were detectable only in slime-producing strains (tested for slime production on Congo Red agar), and never in nonslime-producing ones. This method is a straightforward way of detecting the slime-producing ability by S. epidermidis and S. aureus. In clinical specimens this polymerase chain reaction method enables rapid diagnosis of virulent slime-producing strains with respect to the traditional culture method on Congo Red agar, which requires much more time. Rapid identification of the virulent properties of the bacterial strain responsible for a staphylococcal infection is crucial for deciding treatment.

Abstract: The biocompatibility of two cyanoacrylate surgical glues (Glubran and Glubran 2), supplied by General Enterprise Marketing, Viareggio, Lucca, Italy, was tested through cytotoxicity and blood compatibility tests and the evaluation of antimicrobial activity. Cytotoxicity and blood compatibility tests were performed on the polymerized glues. Using the neutral red uptake test, the extracts from Glubran and Glubran 2 after polymerization were non-toxic to L929 cells only when diluted 1: 10 with culture medium. Glubran and Glubran 2 induced a significant decrease of activated partial thromboplastin time (APTT), which is favourable with regard to the desired haemostasis. The APTT shortening determines a haemostatic effect and therefore contribute to the tissue adhesion induced by the glues. Otherwise, no significant variation of prothrombin activity, fibrinogen, platelet number, total and differential leukocyte count was induced by the glues, which, in addition, did not show haemolytic effect. There was no difference between Glubran and Glubran 2 regarding haemocompatibility. The antimicrobial ability of the unpolymerized glues was tested onto Bacillus subtilis var. niger for 3 weeks: neither Glubran nor Glubran 2 were found effective in this respect. In conclusion, we can assume that cytotoxicity was severe with the undiluted glues, but was acceptable when glues were diluted. On the contrary, blood compatibility was acceptable for the intended use of the glues. No difference was found between Glubran and Glubran 2 after polymerization.

Abstract: Both Staphylococcus epidermidis and Staphylococcus aureus are important causes of infections associated with catheters and other medical devices. It has recently been shown that not only S. epidermidis but also S. aureus can produce slime and carries the ica operon responsible for slime production. In the operon, coexpression of icaA and icaD is required for full slime synthesis. In this study, the presence of icaA and icaD was determined in a collection of 91 staphylococcal (68 S. epidermidis and 23 S. aureus) strains from intravenous catheter-associated infections, in 10 strains from the skin and mucosa of healthy volunteers, and in two reference strains by a PCR method. Slime-forming ability was tested on Congo red agar plates; 49% of S. epidermidis strains from catheters and, surprisingly, 61% of S. aureus strains were icaA and icaD positive and slime forming. All the saprophytic strains turned out to be negative for both icaA and icaD and also non-slime forming. Two S. aureus and one S. epidermidis strain from catheters, detected as icaA and icaD positive by PCR analysis and as slime forming (black colonies) at 24 h on Congo red agar, at 48 h exhibited tiny red spikes at the center of black colonies. The onset of these variants could not be ascribed to a mutagenic potential of Congo red, which, in the Ames test, was devoid of mutagenicity. PCR analysis showed that these red variants were negative for both icaA and icaD and even lacking the entire icaADBC operon. The data reported indicate an important role of ica genes as a virulence marker in staphylococcal infections from intravenous catheters.

Abstract: The aim of this study was to examine the effect of growth conditions on slime production by Staphylococcus aureus clinical isolates. The addition of glucose to the medium enhanced slime production in the majority of Staphylococcus aureus isolates cultured from infections associated with orthopaedic prostheses. Iron limitation also stimulated this ability even in the absence of the additional carbohydrate source. Staphylococcus aureus isolates were classified as Group 1 [strains producing slime only in trypticase soy broth supplemented with 1% glucose (TSBG) or in iron-limited trypticase soy broth (TSB/Fe-)]; Group 2 (slime + only in TSB/Fe-); or Group 3 (slime+ only in TSBG). Seven repeatedly slime-negative strains were stimulated to produce slime by subpassaging in iron-limited medium. Low iron levels, usually found in vivo, could stimulate slime production by Staphylococcus aureus and support chronic infections associated with orthopaedic prostheses.

Abstract: From 50 infected surgical wounds of orthopaedic patients, 43 (86%) staphylococcal strains were isolated. 34 of all these staphylococci belonged to Staphylococcus aureus species (i.e. 68 % of all isolates from surgical wounds; 79% of staphylococcal isolates); 9 were coagulase-negative staphylococci (i.e. 21% of all isolates from surgical wounds; 18% of staphylococcal isolates). Among microorganisms isolated from the wounds we also found 2 (4%) of the Enterobacteriaceae family; 2 (4%) of the Pseudomonas genus; 3 (6%) of the Streptococcus genus. Thus, orthopaedic surgical wounds were infected by staphylococci (mainly S. aureus) more frequently than by other micro-organisms. All the staphylococcal strains were screened for methicillin resistance by agar disk diffusion testing and for the presence of mecA gene responsible for methicillin resistance by PCR. 32% of the S. aureus and 33% of the S. epidermidis strains resulted methicillin resistant and mecA-positive. The data confirm the diffusion of methicillin resistant S. aureus in surgical site infections and shows that the so-called "new pathogens", i.e. S. epidermidis and other coagulase-negative staphylococci, also exhibit a frequent and hazardous methicillin-resisting ability.

Abstract: Enterococcal clinical isolates were investigated for the ability to form biofilm on inert surfaces, as a measure of slime production, in an attempt to find new possible virulence factors for these microorganisms. This property was commonly found among Enterococcus faecalis. Also E. faecium isolates were able to form biofilm, although to a lesser extent; for this species, however, biofilm formation seemed more frequently associated with isolates from infection rather than with environmental strains or isolates from healthy individuals. Biofilm formation was strongly affected by the presence of an additional carbohydrate source in the medium, or by iron deprivation, indicating a role of slime for survival in stressful conditions. Slime-producing E. faecalis were able to survive inside peritoneal macrophages for extended periods compared to slime-negative strains or to slime-positive bacteria grown in conditions depressing slime production. In particular, slime-producing and slime-negative cells showed a decrease of 1 and 2 log units, respectively, at 1 h after infection; slime-negative cells were then rapidly killed, with clearance of bacterial cells at 24 h. Slime-producing bacteria persisted up to 48 h, which was the last time point examined, as after that time viability of both infected and non-infected macrophages started to decline. Scanning electron microscopy observations showed the presence of abundant amorphous extracellular material, of possible polysaccharide nature, embedding bacterial cells to form a multilayered biofilm. Even in conditions not supporting biofilm formation, bacterial cells appeared capsulated, suggesting that capsule and slime might represent different structures. Genes belonging to the epa locus or to a putative icaA homolog did not seem to be involved in synthesis and export of slime.

Abstract: In the last decade an increasing number of research studies have focused on the role of slime formation in Staphylococcus epidermidis and, more recently, also in S. aureus. In this context, much attention is being paid to evaluating the prevalence of slime production among bacteria strains isolated from clinical infections in an attempt to assess the role and the diagnostic value of this well recognised virulence marker. Such types of investigations require reliable techniques to identify slime producing strains. For years, even though based on a subjective chromatic evaluation, the Congo red agar plate (CRA) represented a reference phenotypic test for S. epidermidis. Only with the new introduction of PCR-based techniques, able to specifically identify the genes necessary for slime production, did the accurate genetic classification of slime producing bacteria become possible. In the present investigation, a comparison with new PCR methods confirmed the validity of the classic CRA test, implemented with minor refinements. Thanks to a few modifications it was also possible to adapt and extend the CRA test, making it also suitable to screen S. aureus strains.

Abstract: The blood-contacting properties and the effect on bacterial adhesion of a material based on polyurethane and poly(amido-amine) (PUPA), both in its native form and with the anticoagulant molecules heparin or sulphated hyaluronic acid (HyalS3.5) electrostatically bonded to its surface, were evaluated and compared in vitro. The presence of the biological molecules on the surface was revealed by a dye test and ATR/FTIR analysis. Bound heparin was found to maintain its physiological action, in terms of thrombin inactivation, as well as did free heparin. Moreover, it reduced the degree of platelet adhesion. On the contrary, bound HyalS3.5 lost its anticoagulant activity, though it reduced platelet adhesion. The number of platelets on both modified surfaces was low. Their shape distribution, as determined by SEM, did not differ significantly on the two modified surfaces or with respect to the bare PUPA surface. HyalS3.5 and heparin also inhibited adhesion of Staphylococcus epidermidis to the material. A possible relationship between the platelet and bacterial adhesion is ascribed to the mediating role of plasma proteins.

Abstract: The ability to produce slime and to express a slime-associated antigen was examined in a collection of staphylococcal clinical isolates. Slime-producing strains were found among coagulase-negative staphylococci in percentages comparable to those reported in other studies; surprisingly, a high percentage of Staphylococcus aureus strains also were able to produce this extracellular material. In the latter case, this ability was strongly dependent on the presence of an additional carbohydrate source in the growth medium. Expression of the slime-associated antigen appeared to be species specific and confined to the Staphylococcus epidermidis sensu stricto isolates; its strong association with the ability of these strains to produce thicker biofilms indicated slime-associated antigen as a possible virulence marker for S. epidermidis.

Abstract: The authors evaluated in vitro the adherence of a Staphylococcus epidermidis strain to hydroxyapatite-coated stainless-steel screws-used in orthopaedic surgery for external fracture fixation-in comparison with the adherence to uncoated screws. Evaluations were also performed on analogous groups of screws immersed for 72 and 168 h in a solution at 37 degrees C, in order to simulate the interstitial fluid in a simplified way. Adherence values on coated prostheses resulted significantly lower compared with those observed on metal prostheses, both in basal conditions and after immersion in saline solution. Moreover, both on coated and on uncoated screws a significant reduction in bacterial adherence was noted related to the duration of the prosthesis permanence in saline solution.

Abstract: Several studies have been devoted to identify the adhesion mechanisms of Staphylococcus aureus and Staphylococcus epidermidis, which are the most frequent causes of prosthesis-associated infections. Recently, in particular for Staphylococcus aureus, considerable attention has been given to the host protein receptors as mediators for bacterial adherence. Fibronectin, in important matrix protein, seems to be a major ligand for bacterial adherence in the early stages of infection. To determine the importance of the fibronectin adhesin as virulence factor in Staphylococcus-induced prosthesis infection, a simple and reliable method using a polymerase chain reaction (PCR) was devised to identify fibronectin adhesin genes (fnbA and fnbB). Results obtained by this method were in accordance with those obtained by the in vitro phenotypic characterization of binding ability to fibronectin of Staphylococcus strains.

Abstract: Clinical isolates of Staphylococcus epidermidis are frequently referred to produce a biofilm, known as slime, involved in adherence to medical devices and in resistance to host defences. A high frequency of slime producing Staphylococcus aureus strains was never reported, at least in the case of human isolates. In the present study the production of slime by clinical isolates of S. aureus and S. epidermidis from catheter associated infections and from post-surgical infections was studied by a sensitive method based on culturing the isolates on Congo red agar. The study demonstrates that in nosocomial surgical infections, considered separately from catheter-associated infections, S. aureus emerges as a more prevalent etiologic agent than S. epidermidis, with a proportion of slime producing strains markedly high.

Abstract: Prosthesis-associated infections still represent one of the most serious complications in the clinical use of biomaterials. The most frequent causes are Staphylococcus aureus and Staphylococcus epidermidis. Several studies have been devoted to identify adhesion mechanisms for these bacteria. Slime in particular has been extensively investigated. Recently, in Staphylococcus aureus species, considerable attention has been given to the host protein receptors that have been shown in in vitro assays to serve as substrates for bacterial adhesion. Collagen-rich tissues, as bone and cartilage, that are the preferential sites of staphylococcal infections, are also the tissues that harbour orthopaedic implants. These can be easily coated in vivo by collagen and thus become prone to adhesion of Staphylococci strains which carry the collagen adhesin gene (cna). In this study the frequency of cna was determined within a collection of 35 Staphylococcus aureus strains from orthopaedic prosthesis infections by a PCR method. Also the collagen-binding ability and slime forming capacity was evaluated. 29% of the strains were cna-positive and also able to bind collagen in vitro. 83% of the strains were slime forming. The results indicate that in the examined bacterial population slime-positive strains predominate over the cna-positive strains, with a striking association of the two adhesion mechanisms in cna-positive strains.

Abstract: Deposition and aggregation of lachrymal proteins on the contact lens surface can promote bacterial adherence. Lysozyme is the major tear protein and is also mainly responsible for the formation of protein deposits on contact lenses. Nonsteroidal anti-inflammatory drugs (NSAID) prevent protein aggregation. The effect of a water-soluble NSAID drug on bacterial adherence to high-water-content/ionic disposable contact lenses was examined in a radiolabeling study. Dose-related inhibition of adherence of Staphylococcus aureus, S. epidermidis, and Pseudomonas aeruginosa on both pretreated lenses and after adding the drug to the medium was investigated. When the drug was added to the media, maximal inhibition of S. aureus adherence was observed in trypticase soy broth (59-98% at the lower and higher drug concentrations, respectively); inhibition progressively decreased in calf aqueous humor (48-75%), lysozyme (34-63%), and saline (12-20%) solutions. Inhibition of adherence varied with the three bacterial species; it was maximal with S. aureus, intermediate with S. epidermidis, and minimal with P. aeruginosa. When lenses were pretreated with the drug, consistent, and even higher, inhibitory effects were observed. The results suggest that water-soluble NSAIDs could be used in preventive treatments for conjunctivae and corneal infections in contact lens wearers, and may provide a clue as to which compounds might inhibit protein interaction and bacterial adhesion.

Abstract: Staphylococci are well-recognized pathogens of foreign body-associated infections. The pathogenesis of such infections involves an initial step of contact between the colonizing bacterium and the biomaterial, with subsequent colony formation. Several studies have been devoted to identify adhesion mechanisms for these bacteria. Slime in particular has been extensively investigated. Recently, considerable attention has been given to the host protein receptors that have been shown in in vitro assays to serve as substrates for bacterial adherence. To determine the importance of the collagen adhesin as virulence factor in Staphylococcus-induced prosthesis infection, a simple and reliable method using a polymerase chain reaction (PCR) was devised to identify collagen adhesin gene (cna). By using lysates of ten strains from orthopedic prostheses (5 Staphylococcus aureus and 5 Staphylococcus epidermidis) and two 20-oligonucleotides as primers, a 192-bp region of the cna gene was amplified by PCR and detected by agarose gel electrophoresis. Results obtained by this method were in accordance with those obtained by the in vitro phenotypic characterization of binding ability to collagen of Staphylococcus strains.

Abstract: In an in vitro study, we have quantitatively evaluated the capability of seven different types of silicone to retain a Staphylococcus aureus strain, isolated from a surgical wound. All the silicone specimens were taken from prostheses already used in plastic or ophthalmological surgery. Two polymers were used as controls: polystyrene, because of its known capability to favour in vitro bacterial recovery, and nylon, for its bacterial repellence. The results show that all silicones are suitable substrata for Staphylococcus aureus. However, there are some differences among silicone types. The amounts of bacteria retained from silicone oils are greater than or equal to those obtained from the positive control material.

Abstract: The in vitro biocompatibility of an experimental surface-treated polyurethane was compared with an untreated polyurethane already used for intravascular catheters. The experimental surface was coated with a fluorinated film using a glow discharge treatment. Neither of the catheters was cytotoxic for L929 murine fibroblasts, caused platelet adhesion or release reaction, or changed the mean platelet volume. The surface-treated polyurethane, however, caused a higher adhesion of Staphylococcus aureus than did the untreated one. Therefore, using in vitro testing, it has been ascertained that the examined material, though not being cytotoxic and not modifying platelet behaviour, could favour bacterial adherence.

Abstract: An in vitro quantitative study of the adhesion of a Staphylococcus aureus strain to two types of disposable contact lenses has been carried out. The first type was an ionic/high-water-content (I-HWC) lens (42% Etafilcon A, 58% water) and the second was a non-ionic/low-water-content (Nl-LWC) lens (61.4% poly(2-hydroxyethyl methacrylate), 38.6% water). Adhesion to the two lens types was evaluated both in basic conditions and after treatment with lysozyme. The results showed that I-HWC lenses are more prone to Staphylococcus aureus adhesion than NI-LWC lenses, both untreated (+15.4%) and treated with lysozyme (+20.5%). Lysozyme increased bacterial adhesion by 30.5% on the lenses with lower water content, and by 36.3% on those with higher water content.

Abstract: The haemocompatibility of polyethylene terephthalate (Dacron) coated with pyrolytic carbon was examined in vitro, evaluating its capability of inducing adhesion and platelet activation, and of modifying the intrinsic coagulation pathway. Platelet adhesion was evaluated by counting platelets before and after in vitro contact of human plasma with the material under examination. Platelet activation was evaluated by determining platelet factor 4 (PF4) and thromboxane B2. Intrinsic coagulation pathway alterations were studied by determining activated partial thromboplastin time (APTT) and the activity of single factors. The results obtained show that pyrolytic carbon-coated Dacron induces platelet adhesion, reduction in platelet volume and lower increase in thromboxane production than that obtained after contact with uncoated Dacron. Pyrolytic carbon-coated Dacron does not induce PF4 release, contrary to uncoated Dacron induces a significant release. Moreover, pyrolytic carbon-coated Dacron, induces a significant extension of APTT by reducing the activity of intrinsic pathway factors, particularly factor XI.

Abstract: The Authors evaluated some immunological parameters in women carrying silicone gel-filled breast implants for over one year. Peripheral blood samples from 22 patients were examined in order to assess both the antigenic pattern of lymphocyte subpopulations by cytofluorimetric analysis, and the cell proliferation of PHA-stimulated lymphocytes by the uptake of tritiated thymidine. These tests were performed at the time of the sample withdrawal and after in vitro reexposure to silicone extract for 48-72h. Changes in lymphocyte subpopulations and functional response were observed when patients were divided into groups according to the type of surgery, i.e. breast augmentation or reconstruction, or to the degree of periprosthetic capsular contracture. These results suggest the possibility of an interaction between silicone and the immune system, which cannot be disregarded for the explanation of the silicone related complications.

Abstract: This research aims at evaluating the expression of some adhesive proteins on endothelial cell surface with polyethylene terephthalate coated with pyrolytic carbon (PET + PC). Twenty-two different cultures of human umbilical vein endothelial cells (HUVECs) were put in contact with PET + PC. Both HUVECs grown without the biomaterial and HUVECs incubated with endotoxin were used as control. After 24 h, platelet endothelial cell adhesion molecule-1 (PECAM-1), endothelial leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were evaluated on the cells by monoclonal antibodies and flow cytometry. In agreement with the literature, after 24 h the culture incubated with endotoxin determined a significant increase in the percentage of positive cells for ELAM-1, a significant increase in fluorescence intensity of ICAM-1, and a significant increase in the percentage of positive cells and fluorescence intensity for VCAM-1. After 24 h of culture with PET + PC, no significant variations in the antigens examined were observed. This demonstrates that such material does not activate in vitro the proteins involved in the adhesion between leucocytes and endothelium or in the adhesion between endothelial cells themselves.

Abstract: Several issues concerning the biocompatibility) testing of medical devices as stated in European directive 90/385 and 93/42 are discussed. The authors describe the fundamental characteristics that biomaterials should have to be employed in the human body, the assays useful for testing different aspects of biocompatibility and the harmonized regulations already available or under development. The testing protocol applied by the a. in their Lab covering many issues of biocompatibility is presented: a body of experience was gained over the years and it has been revised under the new regulatory items. In conclusion, the biocompatibility of devices must be assessed following the European harmonized standards, the available. Where proper standards are still lacking, feasible testing methods, even if not officially adopted yet, have to be used.

Abstract: Cell culture systems may be of value in testing the biocompatibility of prosthetic materials before they are introduced into clinical use. In recent years, in vitro methods for assaying biomaterials have gained in importance owing to the growing concern over the use of animals for biomaterials testing. Significant effort is therefore being focused toward developing predictive and quantitative, but also simple and reliable, methods of testing using cultured cells. At present, a number of methods for measuring both the cytotoxicity and the specific cytocompatibility of different materials are available. The usefulness of these systems is no longer confined to screening new materials; they can be used to study the mechanisms of action of various materials during tissue/material interaction. This paper reviews the published literature on the use of cell culture models in evaluating biocompatibility and reports on the personal experience of the authors, who have been using cell culture systems for many years and for different purposes.

Abstract: This quantitative study assessed the in vitro adhesion of Staphylococcus epidermidis on two types of intraocular lenses: conventional poly(methyl methacrylate) (PMMA) and heparin-surface-modified PMMA. Level adhesion was measured by microbiological turbidimetry. We also measured modifications in the bacterium's structural fatty acids after adhesion using gas chromatography.

Abstract: The antibiotic sensitivity of 19 Gram-positive bacterial strains (11 Staphylococcus aureus and 8 Staphylococcus epidermidis) and 16 Gram-negative strains (8 Escherichia coli and 8 Proteus species) was evaluated after contact with stainless steel and with some metals compounding the alloys used for prosthetic devices. The hypothesis was that the resistance to antibiotic therapy of infections associated with prosthetic implants is also due to a modification in the sensitivity of microorganisms. The results, compared to those obtained from control tests, showed only slight variations in the antibiotic sensitivity of the strains put in contact with the metals. In Gram-positive strains, after contact with metals, the increase in sensitivity occurred more frequently than the reduction. In Gram-negative strains, the decrease in sensitivity was more frequent than the increase. Proteus strains showed sensitivity variations more frequently than Escherichia coli strains. Titanium and nickel induced the highest number of variations, both in Gram-positive and Gram-negative strains.

Abstract: The authors have quantitatively evaluated and compared in vitro the Staphylococcus aureus and Staphylococcus epidermidis strain adhesiveness on various types of silicone used in surgery. The results show that there are some adhesiveness differences between the two assayed strains (S. epidermidis adhesiveness > S. aureus adhesiveness) and among silicone types (adhesiveness on soft silicones > adhesiveness on hard silicones). However all silicones are suitable substrata for in vitro bacterial adhesion.

Abstract: An in vitro evaluation was conducted of the adhesion capabilities of rabbit corneal endothelial cells on intraocular lenses (IOLs) made of heparin-coated polymethylmethacrylate (HSM-PMMA). The concave endothelial surfaces of albino rabbit corneas were placed in contact in vitro with the convex surfaces of the optical side of HSM-PMMA IOLs in 4-day cultures. PMMA IOLs served as controls. After an incubation period, the preparations were examined via phase-contrast microscopy and via inverted microscopy after staining with vital dye (neutral red), both with and without the cornea in place. After fixation and staining with Giemsa, the cells adherent on the lens were counted on five different microscopic fields. It was observed that the corneal endothelial cells adhered equally to heparin-coated and untreated PMMA IOLs.

Abstract: Bacterial adhesion on biomaterials is an important cause of associated infection. Many authors have studied the adhesion mechanisms of bacteria on biomaterials. These studies were useful in making materials more and more refractory to bacterial adhesion. We analysed the gas chromatographic modifications of structural fatty acids of a Staphylococcus aureus strain after adhesion on two polymers, poly(methylmethacrylate) (PMMA), whose biological compatibility is known, and heparin-surface-modified PMMA (HSM-PMMA). We noted changes to the chromatographic peaks peculiar to the fatty acids of S. aureus for each tested material and particularly for HSM-PMMA.

Abstract: The authors have examined the alterations in the coagulation pathway of plasma filtered through a polyester filter coated by a hydrophilic polymer. The activation of the coagulation pathway was evaluated by measuring prothrombin time, activated partial thromboplastin time, coagulation factors and fibrinopeptide A. The results obtained demonstrate that the examined filter does not induce significative activation of the coagulation pathway.

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Abstract: The chemico-physical and structural properties and biocompatibility of new polymers used for intraocular lens manufacture are studied. In particular, the vibrational infrared and Raman spectra of fluorinated polymethacrylates are presented and discussed in relation to those of polymethylmethacrylate (PMMA), in order to correlate their structure with their properties and biocompatibility. The thermal analysis points out that the polymers are more stable than polymethylmethacrylate and the biocompatibility tests show that they are as biocompatible as polymethylmethacrylate.

Abstract: Macrophages play an important role at the implant-tissue interface. Their activity is extremely complex but above all they are phagocytic cells. Phagocytosis is accompanied by a sequence of biochemical reactions known as "the oxidative burst" (BO). The fundamental physiological significance of this phenomenon is to provide the phagocytes with an efficient microbicidal mechanism. Our aim was to prepare an objective method for screening biomaterials that could influence macrophagic defence mechanisms against a possible implant contamination.

Abstract: Among the conditions that a material must satisfy to be considered biocompatible, most important is the non negative influence on the cellular viability of the surrounding tissue (cytocompatibility). The aim of our work was to evaluate in vitro, whether different types of materials (usually used in surgery), alter the viability parameters or the morphological characteristics of the cultured cells in contact with them.

Abstract: In surgical divisions, where you can find the highest percentage of hospital infections, the surgical wounds are definitively the most frequent localization. The aim of our study was to evaluate whether bacterial-biomaterial interactions could influence the growth of infections in sutured tissues. We prepared a method for in vitro bacterial adhesiveness testing based on turbidimetric analysis. The results obtained show a different grade of bacterial adhesion in relation to physical and chemical characteristics of the material examined.