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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.