« Les biomatériaux inhibiteurs de l'adhérence et de la prolifération bactérienne : un enjeu pour la prévention des infections sur matériel prothétique »

Bacterial infections on implanted prosthetic materials represent one of the major problems of public health and one of the first causes of nosocomial infections. Infections on implanted devices are extremely resistant to antibiotics as well as to host defences and frequently persist until the removal of the implant which remains the standard therapy. In order to prevent infection, several strategies are proposed and/or developed; amongst them heparin or antibiotic impregnations were extensively studied and gave interesting results nevertheless the most promising solutions consists in synthesizing and/or grafting bioactive polymers able to inhibit bacterial adhesion.     

Inhibition of bacterial and leukocyte adhesion under shear stress conditions by material surface chemistry

Biomaterial-centered infections, initiated by bacterial adhesion, persist due to a compromised host immune response. Altering implant materials with surface modifying endgroups (SMEs) may enhance their biocompatibility by reducing bacterial and inflammatory cell adhesion. A rotating disc model, which generates shear stress within physiological ranges, was used to characterize adhesion of leukocytes and Staphylococcus epidermidis on polycarbonate-urethanes and polyetherurethanes modified with SMEs (polyethylene oxide, fluorocarbon and dimethylsiloxane) under dynamic flow conditions. Bacterial adhesion in the absence of serum was found to be mediated by shear stress and surface chemistry, with reduced adhesion exhibited on materials modified with polydimethylsiloxane and polyethylene oxide SMEs. In contrast, bacterial adhesion was enhanced on materials modified with fluorocarbon SMEs. In the presence of serum, bacterial adhesion was primarily neither material nor shear dependent. However, bacterial adhesion in serum was significantly reduced to 10% compared to adhesion in serum-free media. Leukocyte adhesion in serum exhibited a shear dependency with increased adhesion occurring in regions exposed to lower shear-stress levels of 7 dyne/cm². Additionally, polydimethylsiloxane and polyethylene oxide SMEs reduced leukocyte adhesion on polyether-urethanes. In conclusion, these results suggest that surface chemistry and shear stress can mediate bacterial and cellular adhesion. Furthermore, materials modified with polyethylene oxide SMEs are capable of inhibiting bacterial adhesion, consequently minimizing the probability of biomaterial-centered infections.     

PHACOS,a functionalized bacterial polyester with bactericidal activity against methicillin-resistant Staphylococcus aureus

Biomaterial-associated infections represent a significant clinical problem, and treatment of these microbial infections is becoming troublesome due to the increasing number of antibiotic-resistant strains. Here, we report a naturally functionalized bacterial polyhydroxyalkanoate (PHACOS) with antibacterial properties. We demonstrate that PHACOS selectively and efficiently inhibits the growth of methicillin-resistant Staphylococcus aureus (MRSA) both in vitro and in vivo. This ability has been ascribed to the functionalized side chains containing thioester groups. Significantly less (3.2-fold) biofilm formation of S. aureus was detected on PHACOS compared to biofilms formed on control poly(3-hydroxyoctanoate-co-hydroxyhexanoate) and poly(ethylene terephthalate), but no differences were observed in bacterial adhesion among these polymers. PHACOS elicited minimal cytotoxic and inflammatory effects on murine macrophages and supported normal fibroblast adhesion. In vivo fluorescence imaging demonstrated minimal inflammation and excellent antibacterial activity for PHACOS compared to controls in an in vivo model of implant-associated infection. Additionally, reductions in neutrophils and macrophages in the vicinity of sterile PHACOS compared to sterile PHO implant were observed by immunohistochemistry. Moreover, a similar percentage of inflammatory cells was found in the tissue surrounding sterile PHACOS and S. aureus pre-colonized PHACOS implants, and these levels were significantly lower than S. aureus pre-colonized control polymers. These findings support a contact active surface mode of antibacterial action for PHACOS and establish this functionalized polyhydroxyalkanoate as an infection-resistant biomaterial.     

Hydrodynamic Shear and Collagen Receptor Density Determine the Adhesion Capacity of S. aureus to Collagen

Dynamic bacterial adhesion has recently gained significant attention due to its role in the initiation of infectious diseases. Staphylococcus aureus binding to collagen has been shown to be an important event in the pathogenesis of infection. Staphylococcal strains have exhibited wide variability in their level of collagen binding, which may be a result of the collagen receptor expression level. In this study, the dynamic adhesion to collagen for several S. aureus strains was quantified at physiological wall shear rates in a parallel-plate flow chamber. In addition, the collagen receptor density was quantified for each strain. An existing theoretical framework was used to analyze the dependence of adhesion on receptor density. Intrinsic kinetic adhesion parameters were determined and demonstrated to be strong functions of receptor density for all strains. These results suggest that staphylococcal adhesion to collagen is heavily dependent on the receptor density. Using this analytical approach it is possible to predict the dynamic adhesion of S. aureus to collagen in vitro by only measuring the collagen receptor density. © 2001 Biomedical Engineering Society. PAC01: 8717-d, 8714Ee, 8719Xx, 8719Tt     

Inhibition of Staphylococcus epidermidis adhesion on titanium surface with bioactive water-soluble copolymers bearing sulfonate, phosphate or carboxylate functions

Implanted prostheses are sometimes subject to bacterial infections, which can threat their benefit rule on a long-term basis. Various methods are studied to fight against these infections. Among them, the grafting of bioactive polymers onto the prosthesis surface shows up as a promising way to the problem of infections. This work presents the influence of various water-soluble bioactive polymers on the inhibition of the Staphylococcus epidermidis adhesion on the titanium samples surfaces initially preadsorbed with various proteins. Whatever the studied protein is, it is shown that the bioactive polymer containing sulfonate functions generates an inhibition of the adhesion of Staphylococcus epidermidis. For a plasma preadsorption, the inhibition rate rises up to 68% when the concentration of sulfonate function is 2.5μmol/L. Titanium surfaces grafted with the bioactive polymer were also tested. We find an inhibitive activity of the adhesion close to that of the previous case. These preliminary results can point up a clinical interest in the fight against the medical devices infection, because they highlight a clear local effect of S. epidermidis adhesion inhibition. Copolymers containing other functional groups (phosphate or carboxylate) were dissolved in a bacterial suspension to monitor the influence of the composition on the adhesion inhibition. Their inhibition rates are not significantly lower than those of pNaSS homopolymers, as much as the sulfonate function proportion remains higher than 50%. Thus, the sulfonate function is the main responsible for the inhibition of the S. epidermidis adhesion.     

Neutrophils in innate host defense against <Emphasis Type="BoldItalic">Staphylococcus aureus</Emphasis> infections

Staphylococcus aureus has been an important human pathogen throughout history and is currently a leading cause of bacterial infections worldwide. S. aureus has the unique ability to cause a continuum of diseases, ranging from minor skin infections to fatal necrotizing pneumonia. Moreover, the emergence of highly virulent, drug-resistant strains such as methicillin-resistant S. aureus in both healthcare and community settings is a major therapeutic concern. Neutrophils are the most prominent cellular component of the innate immune system and provide an essential primary defense against bacterial pathogens such as S. aureus. Neutrophils are rapidly recruited to sites of infection where they bind and ingest invading S. aureus, and this process triggers potent oxidative and non-oxidative antimicrobial killing mechanisms that serve to limit pathogen survival and dissemination. S. aureus has evolved numerous mechanisms to evade host defense strategies employed by neutrophils, including the ability to modulate normal neutrophil turnover, a process critical to the resolution of acute inflammation. Here we provide an overview of the role of neutrophils in host defense against bacterial pathogens and discuss strategies employed by S. aureus to circumvent neutrophil function.     

Staphylococcal Presence Alters Thrombus Formation Under Physiological Shear Conditions in Whole Blood Studies

The ability of platelets to aggregate at a site of injury and form a thrombus is central to normal hemostasis and host survival. Staphylococcus aureus is a major pathogenic organism that has been described as interacting with platelets and promoting platelet aggregation. However, the fundamental effects of bacterial presence on thrombus formation are poorly understood. In this study, we quantify thrombus formation in whole blood in the presence and absence of S. aureus under physiological shear conditions. Using confocal microscopy we observed that S. aureus causes a reduction in thrombus volumes and alters the morphological structure of the growing thrombi.     

Antiadhesive and antibiofilm activity of hyaluronic acid against bacteria responsible for respiratory tract infections

To address the problem of limited efficacy of existing antibiotics in the treatment of bacterial biofilm, it is necessary to find alternative remedies. One candidate could be hyaluronic acid; this study therefore aimed to evaluate the in vitro antiadhesive and antibiofilm activity of hyaluronic acid toward bacterial species commonly isolated from respiratory infections. Interference exerted on bacterial adhesion was evaluated by using Hep‐2 cells, while the antibiofilm activity was assessed by means of spectrophotometry after incubation of biofilm with hyaluronic acid and staining with crystal violet. Our data suggest that hyaluronic acid is able to interfere with bacterial adhesion to a cellular substrate in a concentration‐dependent manner, being notably active when assessed as pure substance. Moreover, we found that Staphylococcus aureus biofilm was more sensitive to the action of hyaluronic acid than biofilm produced by Haemophilus influenzae and Moraxella catarrhalis. In conclusion, hyaluronic acid is characterized by notable antiadhesive properties, while it shows a moderate activity against bacterial biofilm. As bacterial adhesion to oral cells is the first step for colonization, these results further sustain the role of hyaluronic acid in prevention of respiratory infections.     

Mast cells are activated by Staphylococcus aureus in vitro but do not influence the outcome of intraperitoneal S. aureus infection in vivo

Staphylococcus aureus is a major pathogen that can cause a broad spectrum of serious infections including skin infections, pneumonia and sepsis. Peritoneal mast cells have been implicated in the host response towards various bacterial insults and to provide mechanistic insight into the role of mast cells in intraperitoneal bacterial infection we here studied the global effects of S. aureus on mast cell gene expression. After co‐culture of peritoneal mast cells with live S. aureus we found by gene array analysis that they up‐regulate a number of genes. Many of these corresponded to pro‐inflammatory cytokines, including interleukin‐3, interleukin‐13 and tumour necrosis factor‐α. The cytokine induction in response to S. aureus was confirmed by ELISA. To study the role of peritoneal mast cells during in vivo infection with S. aureus we used newly developed Mcpt5‐Cre⁺ × R‐DTA mice in which mast cell deficiency is independent of c‐Kit. This is in contrast to previous studies in which an impact of mast cells on bacterial infection has been proposed based on the use of mice whose mast cell deficiency is a consequence of defective c‐Kit signalling. Staphylococcus aureus was injected intraperitoneally into mast‐cell‐deficient Mcpt5‐Cre⁺ × R‐DTA mice using littermate mast‐cell‐sufficient mice as controls. We did not observe any difference between mast‐cell‐deficient and control mice with regard to weight loss, bacterial clearance, inflammation or cytokine production. We conclude that, despite peritoneal mast cells being activated by S. aureus in vitro, they do not influence the in vivo manifestations of intraperitoneal S. aureus infection.     

Differential roles of sortase-anchored surface proteins and wall teichoic acid in Staphylococcus aureus nasal colonization

Most of the severe bacterial infections originate from the endogenous microflora of human body surfaces. However, the molecular basis of colonization, e.g. of the human nose by Staphylococcus aureus, has remained incompletely understood. Several surface-exposed proteins and wall teichoic acid (WTA) polymers have previously been implicated in S. aureus attachment to nasal epithelial cells. Here we dissect the role of these molecules in colonization using S. aureus sortase A (srtA) and tagO mutants deficient in surface protein and WTA display, respectively. Although the two mutants were similarly affected in attachment to nasal cells they were abrogated in binding to different types of epithelial ligands. Surface protein sorting, but not WTA, were required for keratin- or fibronectin-mediated interactions while only WTA-mediated binding to nasal cells was effectively inhibited by polyinosinic acid, indicating a possible role of scavenger receptor-like molecules in WTA-dependent epithelial interactions. Both mutants exhibited profound colonization defects in a cotton rat nasal colonization model, albeit at different stages of colonization (>90% reduced bacterial counts at 24 h or several days after inoculation with the tagO or srtA mutant, respectively). These data indicate that S. aureus nasal colonization is a multifactorial process with various ligands affecting initial colonization and prolonged persistence in different ways. Our studies should be useful in the development of new preventive and therapeutic strategies.     

Exploring <Emphasis Type="BoldItalic">Staphylococcus aureus</Emphasis> pathways to disease for vaccine development

Staphylococcus aureus is a commensal of the human skin or nares and a pathogen that frequently causes skin and soft tissue infections as well as bacteremia and sepsis. Recent efforts in understanding the molecular mechanisms of pathogenesis revealed key virulence strategies of S. aureus in host tissues: bacterial scavenging of iron, induction of coagulation pathways to promote staphylococcal agglutination in the vasculature, and suppression of innate and adaptive immune responses. Advances in all three areas have been explored for opportunities in vaccine design in an effort to identify the critical protective antigens of S. aureus. Human clinical trials with specific subunit vaccines have failed, yet provide important insights for the design of future trials that must address the current epidemic of S. aureus infections with drug-resistant isolates (MRSA, methicillin-resistant S. aureus).     

Metal ion acquisition in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>: overcoming nutritional immunity

Transition metals are essential nutrients to virtually all forms of life, including bacterial pathogens. In Staphylococcus aureus, metal ions participate in diverse biochemical processes such as metabolism, DNA synthesis, regulation of virulence factors, and defense against oxidative stress. As an innate immune response to bacterial infection, vertebrate hosts sequester transition metals in a process that has been termed “nutritional immunity.” To successfully infect vertebrates, S. aureus must overcome host sequestration of these critical nutrients. The objective of this review is to outline the current knowledge of staphylococcal metal ion acquisition systems, as well as to define the host mechanisms of nutritional immunity during staphylococcal infection.     

Quantification of initial adhesion of Enterococcus faecalis to medical grade polymers using a DNA-based fluorescence assay

This paper reports on the use of a DNA-based fluorescence assay to study and quantify the initial interactions of the uropathogen Enterococcus faecalis with different polymers commonly used for the fabrication of medical devices and implants, including polyurethane (PU), silicone (SI), high-density polyethylene (HDPE), polyamide (PA), poly(methyl methacrylate) (PMMA) and polytetrafluoroethylene (PTFE). To follow the kinetics of E. faecalis adhesion, polymer samples were incubated in bacterial solution for various times and the relative concentration of adhered bacteria was obtained using two methods: commonly used CFU plate counting and a DNA quantification assay. Results obtained from DNA-based fluorescence assays showed that E. faecalis adhesion on PU is 3-times higher than that on PTFE following exposure to bacteria for 180 min. Neither surface wettability nor surface roughness of the studied polymers was found to correlate with E. faecalis adhesion, suggesting the involvement of much more complex adhesion mechanisms of bacteria onto surfaces. SEM micrographs of adhered bacteria illustrated that adhesion was different depending on the type of polymeric substrate: adhesion on PU samples was characterized by the aggregation of bacterial cells in dense clusters, as well as by the presence of fimbriae between cells and the substrate, which could explain the high adhesion to PU compared to the other polymers. This work demonstrated that the bacterial adhesion to polymers occurs at an early stage of the contact and suggests that the initial adhesion stage should be controlled, in order to prevent subsequent biofilm formation and, thus, reduce the risk of implant-associated infections.     

Acceleration of the direct identification of Staphylococcus aureus versus coagulase-negative staphylococci from blood culture material: a comparison of six bacterial DNA extraction methods

To accelerate differentiation between Staphylococcus aureus and coagulase-negative staphylococci (CNS), this study aimed to compare six different DNA extraction methods from two commonly used blood culture materials, i.e. BACTEC and BacT/ALERT. Furthermore, we analysed the effect of reduced blood culture incubation for the detection of staphylococci directly from blood culture material. A real-time polymerase chain reaction (PCR) duplex assay was used to compare the six different DNA isolation protocols on two different blood culture systems. Negative blood culture material was spiked with methicillin-resistant S. aureus (MRSA). Bacterial DNA was isolated with automated extractor easyMAG (three protocols), automated extractor MagNA Pure LC (LC Microbiology Kit M^Grade), a manual kit MolYsis Plus and a combination of MolYsis Plus and the easyMAG. The most optimal isolation method was used to evaluate reduced bacterial incubation times. Bacterial DNA isolation with the MolYsis Plus kit in combination with the specific B protocol on the easyMAG resulted in the most sensitive detection of S. aureus, with a detection limit of 10 CFU/ml, in BacT/ALERT material, whereas using BACTEC resulted in a detection limit of 100 CFU/ml. An initial S. aureus or CNS load of 1 CFU/ml blood can be detected after 5 h of incubation in BacT/ALERT 3D by combining the sensitive isolation method and the tuf LightCycler assay.     

Monoclonal antibody against l-lectin module of SraP blocks adhesion and protects mice against Staphylococcus aureus challenge

Background/purposeSraP is a serine-rich repeat protein (SRRP) from Staphylococcus aureus that binds to sialylated receptors to promote bacterial adhesion to and invasion into host epithelial cells, mediated by the l-lectin module of its ligand-binding region.MethodsThe sequence encoding the L-lectin module of SraP was inserted into pET28a plasmid, and the recombinant protein was purified by His label affinity chromatography. A monoclonal antibody (mAb) against the l-lectin module was obtained and confirmed by enzyme-linked immunosorbent assay and western blotting. The effect of the mAb on S. aureus adhesion and invasion was assessed in A₅₄₉ cells and mice subjected to S. aureus challenge.ResultsWe successfully obtained a mAb against the l-lectin module of SraP. Pre-incubation with the mAb dramatically inhibited the bacteria's ability to adhere to and invade A₅₄₉ cells. Moreover, mice administered mAb through tail vein injection had significantly fewer bacteria in the blood.ConclusionThe anti-SraP_L-Lectin mAb significantly reduced the adherence and invasion of S. aureus to host cells. This study lays the foundation for the future development of the l-lectin module of SraP as a target for the prevention and treatment of S. aureus infection. Our findings suggest that specific subdomains of SRRPs may represent potential antibacterial drug targets for intervention.     

Effect of surface roughness and sterilization on bacterial adherence to ultra-high molecular weight polyethylene

Sterilization with ethylene oxide (EO) and gas plasma (GP) are well-known methods applied to ultra-high molecular weight polyethylene (UHMWPE) surfaces in the belief that they prevent major material changes caused by gamma irradiation. However, the influence of these surface sterilization methods on bacterial adherence to UHMWPE is unknown. UHMWPE samples with various degrees of roughness (0.3, 0.8 and 2.0 µm) were sterilized with either GP or EO. The variations in hydrophobicity, surface free energy and surface functional groups were investigated before and after sterilization. Sterilized samples were incubated with either Staphylococcus aureus or Staphylococcus epidermidis in order to study bacterial adherence to these materials. Fewer bacteria adhered to UHMWPE after sterilization with EO than after sterilization with GP, especially to the smoothest surfaces. No changes in chemical composition of the UHMWPE surface due to sterilization were observed using X-ray photoemission spectroscopy analysis. The decreased bacterial adherence to UHMWPE found at the smoothest surfaces after sterilization with EO was not directly related to changes in chemical composition. Increased bacterial adherence to rougher surfaces was associated with increased polar surface energy of EO-sterilized surfaces.     

Association betweenStaphylococcus aureus nasopharyngeal colonization and septicemia in patients infected with the human immunodeficiency virus

In an attempt to identify risk factors forStaphylococcus aureus septicemia, 136 consecutive HIV-infected patients were investigated for the presence of nasopharyngeal colonization withStaphylococcus aureus and subsequentStaphylococcus aureus infection. Sixty of 136 (44.1 %) HIV-infected patients had staphylococci which were detected in the nasopharynx on initial culture compared to 12 of 39 (30.8 %) patients with chronic diseases and 11 of 47 (23.4 %) healthy hospital staff. Another 12 HIV-infected subjects proved to beStaphylococcus aureus carriers on follow-up cultures. Patients with full-blown AIDS had a higher carriage rate compared to subjects who were only HIV-positive (p<0.05), indicating thatStaphylococcus aureus colonized patients were more severely ill. Eight patients withStaphylococcus aureus septicemia were observed, all of whom were carriers; no septicemia occurred in the non-colonized patients (p<0.01). Colonized patients with neutropenia (< 1000/µl) were significantly more likely to develop septicemia (p<0.01). Nasopharyngeal colonization withStaphylococcus aureus and the presence of an indwelling catheter were established to be factors that help identify patients at risk of acquiring subsequentStaphylococcus aureus infection.     

Inhibition of bacterial and leukocyte adhesion under shear stress conditions by material surface chemistry

Biomaterial-centered infections, initiated by bacterial adhesion, persist due to a compromised host immune response. Altering implant materials with surface modifying endgroups (SMEs) may enhance their biocompatibility by reducing bacterial and inflammatory cell adhesion. A rotating disc model, which generates shear stress within physiological ranges, was used to characterize adhesion of leukocytes and Staphylococcus epidermidis on polycarbonate-urethanes and polyetherurethanes modified with SMEs (polyethylene oxide, fluorocarbon and dimethylsiloxane) under dynamic flow conditions. Bacterial adhesion in the absence of serum was found to be mediated by shear stress and surface chemistry, with reduced adhesion exhibited on materials modified with polydimethylsiloxane and polyethylene oxide SMEs. In contrast, bacterial adhesion was enhanced on materials modified with fluorocarbon SMEs. In the presence of serum, bacterial adhesion was primarily neither material nor shear dependent. However, bacterial adhesion in serum was significantly reduced to < or = 10% compared to adhesion in serum-free media. Leukocyte adhesion in serum exhibited a shear dependency with increased adhesion occurring in regions exposed to lower shear-stress levels of < or = 7 dyne/cm2. Additionally, polydimethylsiloxane and polyethylene oxide SMEs reduced leukocyte adhesion on polyether-urethanes. In conclusion, these results suggest that surface chemistry and shear stress can mediate bacterial and cellular adhesion. Furthermore, materials modified with polyethylene oxide SMEs are capable of inhibiting bacterial adhesion, consequently minimizing the probability of biomaterial-centered infections.