Ilaria Cangini

Scenery of Staphylococcus implant infections in orthopedics

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

The presence of both bone sialoprotein-binding protein gene and collagen adhesin gene as a typical virulence trait of the major epidemic cluster in isolates from orthopedic implant infections

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.

Biofilm extracellular-DNA in 55 Staphylococcus epidermidis clinical isolates from implant infections.

Biofilm formation is broadly recognized as an important virulence factor in many bacterial species implicated in implant-related opportunistic infections. In spite of a long history of research and many investigative efforts aimed at elucidating their chemical composition, structure, and function, the nature of bacterial biofilms still remains only partly revealed. Over the years, different extracellular polymeric substances (EPS) have been described that contribute functionally and structurally to the organization of biofilms. Recently extracellular DNA (eDNA) has emerged as a quantitatively conspicuous and potentially relevant structural component of microbial biofilms of many microbial species, Staphylococcus aureus and S. epidermidis among them. The present study aims at comparatively investigating the amount of eDNA present in the biofilm of 55 clinical isolates of S. epidermidis from postsurgical and biomaterial-related orthopedic infections. Quantification of eDNA was performed by a non-destructive method directly on bacterial biofilms formed under static conditions on the plastic surface of 96-well plates.

Bacterial adhesion to poly-(D,L)lactic acid blended with vitamin E: toward gentle anti-infective biomaterials.

Anti-infective properties of biomedical materials are often achieved by loading or coating them with powerful bactericides. Undesirably, these bioactive molecules can damage the host cells at the biomaterial-tissues interface and, sometimes, even determine systemic toxic effects. The search for biomaterials able to actively resist infection while displaying a safe cytocompatibility profile toward eukaryotic cells is being progressively developed. Poly-(D,L)lactic acid (PLA) is a broadly used resorbable material with established biocompatibility properties. The dissolving surfaces of a biodegradable material tend to be per se elusive for bacteria. Here, films of pristine PLA, of PLA blended with vitamin E (VitE) and PLA blended with vitamin E acetate (VitE ac) were challenged in vitro with the biofilm-producers Staphylococcus epidermidis RP62A and Staphylococcus aureus ATCC25923. The bacterial adhesion properties of the different materials were investigated on small film disc specimens by a method based on microtiter plates. Adherent bacteria were quantified by both CFU plating and bioluminescence. Significant decrease in bacterial adhesion and biofilm accumulation was found on the surface of both the enriched polymers. These findings, together with the favorable intrinsic properties of PLA and the desirable bioactivities conferred by VitE, point up the VitE-blended PLA polymers as gentle anti-infective biomaterials.

Exopolysaccharide production by Staphylococcus epidermidis and its relationship with biofilm extracellular DNA

Implant-related infections are difficult to treat because they are very often associated with biofilm-forming micro-organisms capable of resisting host immune defenses and surviving conventional antibiotic treatments. In Staphylococcus epidermidis biofilm-forming strains, the polysaccharide intercellular adhesin (PIA), whose expression is encoded by the icaADBC operon, is recognized as a main staphylococcal accumulation mechanism. Nevertheless, various observations have shown that PIA expression is dispensable and a variety of additional/alternative accumulation mechanisms, including extracellular DNA (eDNA) and several other factors of proteic nature, can compensate for icaADBC low expression or even for its absence. A suggestive hypothesis points to the possibility that changes in biofilm extracellular matrix composition can be induced in different environmental niches. In this study we aimed at investigating the relationship between the exopolysaccharide and eDNA biofilm components, screening 55 S. epidermidis clinical isolates by means of a simple fluorescence-based microtiter-plate assay. Our findings indicate the existence of a certain degree of correlation, although not a strict one, between eDNA and the exopolysaccharide component. The presence of exopolysaccharide greatly varied even in strains belonging to the same strain type determined by automated riboprinting.

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

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

Polymorphisms of agr locus correspond to distinct genetic patterns of virulence in Staphylococcus aureus clinical isolates from orthopedic implant infections.

Staphylococcus aureus is the leading etiologic agent of orthopedic implant infections. It is endowed with the accessory gene regulator (agr) locus that modulates expression of many virulence genes. Four allelic groups of agr have been recognized within this bacterial species. Here, 200 S. aureus isolates from orthopedic implant infections, typed at the start depending on their agr group, were screened for the presence of adhesin and leukotoxin genes. Interestingly, specific virulence gene patterns emerged in association with agr groups. The most frequently observed agr groups, agr I and agr II, were associated with the presence of sdrE, fib (agr II more than agr I), fnbB (agr I more than agr II), and lukE/lukD (agr II more than agr I). The third more frequent agr group, agr III, differed clearly from agr I and II, exhibiting high prevalence of bbp, generally not harbored by agr I and II, and copresence of bbp with cna, whereas high prevalence of the tandem sdrE/fib marked definitely agr II (91% of agr II isolates), and, though less strictly, agr I, in which prevailed the peculiar fib/fnbB pattern. The only four isolates belonging to agr IV showed full copresence of bbp with fib. Results point out distinct patterns of virulence genes, which underlie distinct evolutive strategies associated to agr groups in S. aureus causing orthopedic implant infections.

An overview of the methodological approach to the in vitro study of anti-infective biomaterials

Biomaterial-associated infections have an enormous impact in terms of morbidity of the patients and costs to national health systems. Perioperative antibiotics and aseptic procedures have not proved sufficient to eradicate the occurrence of this type of infections which often lead to devastating effects. Adjunctive strategies for preventing the establishment of infections are increasingly being centered on the development of new biomaterials with anti-infective properties. The creation of new anti-infective biomaterials can be obtained by alternative approaches oriented to achieve either bacteria-repellent surfaces or bioactive surfaces expressing self-sterilizing properties when not even able to treat preexisting infections in the surrounding tissues. Here, we offer a short overview of the currently available in vitro methods that can be used to investigate and assess the performance of anti-infective biomaterials, with special emphasis on those whose mechanism of action is based on bacteria-repellent surfaces.

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

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

Vitamin E modifies poly(D,L)lactic acid wettability and reduces bacterial adhesion

Highly biocompatible polylactic acid (PLA)-derived polymers are used for different biomedical applications such as orthopaedic screws and drug delivery devices. Nevertheless their clinical use is limited by their proinflammatory characteristics. Vitamin E (α-tocopherol, Vit. E), a natural antioxidant and anti-inflammatory agent has been used to improve different biomaterials biostability [1], and among them also P(D,L)LA [2]. In this work, addition of Vit.E (10-40% w/v) to P(D,L)LA films obtained by solvent casting technique increased polymer surface wettability and human plasma protein adsorption, while addition of Vit.E acetate (Vit.E Ac, 10-40% w/v), the acetic ester of α-tocopherol, often used as an alternative to Vit.E itself, failed in modifying polymer wettability. On the other hand, bacterial adhesion experiments onto control, Vit.E and Vit.E Ac enriched P(D,L)LA films showed that both presence of Vit.E and Vit.E Ac was able to reduce the adhesion of the RP62A Staphylococcus epidermidis strain [3]. In particular, in PLA + Vit. E samples the decrease in bacterial adhesion was of 56%, while, in the case of PLA + Vit. E Ac samples the decrease was of 40%. These preliminary data suggest that Vit. E addition to PLA containing medical devices could improve their resistance to bacterial infections.