Lucio Montanaro

A review of the biomaterials technologies for infection-resistant surfaces.

Anti-infective biomaterials need to be tailored according to the specific clinical application. All their properties have to be tuned to achieve the best anti-infective performance together with safe biocompatibility and appropriate tissue interactions. Innovative technologies are developing new biomaterials and surfaces endowed with anti-infective properties, relying either on antifouling, or bactericidal, or antibiofilm activities. This review aims at thoroughly surveying the numerous classes of antibacterial biomaterials and the underlying strategies behind them. Bacteria repelling and antiadhesive surfaces, materials with intrinsic antibacterial properties, antibacterial coatings, nanostructured materials, and molecules interfering with bacterial biofilm are considered. Among the new strategies, the use of phages or of antisense peptide nucleic acids are discussed, as well as the possibility to modulate the local immune response by active cytokines. Overall, there is a wealth of technical solutions to contrast the establishment of an implant infection. Many of them exhibit a great potential in preclinical models. The lack of well-structured prospective multicenter clinical trials hinders the achievement of conclusive data on the efficacy and comparative performance of anti-infective biomaterials.

Biofilm in implant infections: its production and regulation.

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 β-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.

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

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

Presence of icaA and icaD Genes and Slime Production in a Collection of Staphylococcal Strains from Catheter-Associated Infections

ABSTRACT Both Staphylococcus epidermidis andStaphylococcus 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 oficaA and icaD is required for full slime synthesis. In this study, the presence of icaA andicaD 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 wereicaA and icaD positive and slime forming. All the saprophytic strains turned out to be negative for bothicaA and icaD and also non-slime forming. TwoS. 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.

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

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

Scenery of Staphylococcus implant infections in orthopedics

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

Cytotoxicity, blood compatibility and antimicrobial activity of two cyanoacrylate glues for surgical use

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.

Etiology of implant orthopedic infections : A survey on 1027 clinical isolates

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.

A review of the clinical implications of anti-infective biomaterials and infection-resistant surfaces.

Infection is currently regarded as the most severe and devastating complication associated to the use of biomaterials. The important social, clinical and economic impacts of implant-related infections are promoting the efforts to obviate these severe diseases. In this context, the development of anti-infective biomaterials and of infection-resistant surfaces is being regarded as the main strategy to prevent the establishment of implant colonisation and biofilm formation by bacteria. In this review, the attention is focused on the biomaterial-associated infections, from which the need for anti-infective biomaterials originates. Biomaterial-associated infections differ markedly for epidemiology, aetiology and severity, depending mainly on the anatomic site, on the time of biomaterial application, and on the depth of the tissues harbouring the prosthesis. Here, the diversity and complexity of the different scenarios where medical devices are currently utilised are explored, providing an overview of the emblematic applicative fields and of the requirements for anti-infective biomaterials.

Detection of slime production by means of an optimised Congo red agar plate test based on a colourimetric scale in Staphylococcus epidermidis clinical isolates genotyped for ica locus.

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.