RESUMO
Biomaterial-associated infections (BAIs) are frequent complications in the use of medical devices (biomaterials) correlated with considerable patient discomfort and high treatment costs. The presence of a biomaterial in the host causes derangement of local immune responses increasing susceptibility to infection. Dendritic cells (DCs) have an important role in directing the nature of immune responses by activating and controlling CD4+ T helper (Th) cell responses. To assess the immunomodulatory effect of the combined presence of biomaterials and Staphylococcus aureus (S. aureus) or Staphylococcus epidermidis (S. epidermidis), DC-mediated T cell proliferation and Th1/Th2 cell development were measured using an in vitro human cell system. Poly(trimethylene carbonate) (PTMC) and poly(D,L-lactic acid) (PDLLA) modified the production of the DC pro-inflammatory cytokines TNF-α, IL-6 and IL-23 in response to S. aureus and S. epidermidis. However, this modified cytokine production did not cause differences in Th1/Th2 cell polarisation, showing a Th1 cell predominance. In the absence of staphylococci, neither of the biomaterials induced DC-mediated T cell proliferation or Th1/Th2 cell polarisation. Moreover, either in the absence or presence of the biomaterials, S. aureus was a more potent inducer of DC cytokine secretion, T cell proliferation and Th1 cell development than S. epidermidis. In conclusion, although PTMC and PDLLA modulated DC cytokine responses to staphylococci, this did not alter the resulting Th cell development. This result suggested that, in this human cell model, Th1/Th2 cell responses were mainly determined by the species of bacteria and that PTMC or PDLLA did not detectably influence these responses.
Assuntos
Células Dendríticas/microbiologia , Dioxanos/farmacologia , Poliésteres/farmacologia , Polímeros/farmacologia , Staphylococcus/fisiologia , Células Th1/citologia , Células Th2/citologia , Materiais Biocompatíveis/farmacologia , Biomarcadores/metabolismo , Polaridade Celular , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Humanos , Células Th1/efeitos dos fármacos , Células Th2/efeitos dos fármacosRESUMO
Prevention of biomaterial-associated infections (BAI) remains a challenging problem, in particular due to the increased risk of resistance development with the current antibiotic-based strategies. Metallic orthopaedic devices, such as non-cemented implants, are often inserted under high mechanical stress. These non-cemented implants cannot be protected by e.g. antibioticreleasing bone cement or other antimicrobial approaches, such as the use of bioactive glass. Therefore, in order to avoid abrasion during implantation procedures, we developed an antimicrobial coating with great mechanical stability for orthopaedic implants, to prevent Staphylococcus aureus BAI. We incorporated 5 and 10 wt % chlorhexidine in a novel mechanically stable epoxy-based coating, designated CHX5 and CHX10, respectively. The coatings displayed potent bactericidal activity in vitro against S. aureus, with over 80 % of the release (19 µg/cm2 for CHX5 and 41 µg/cm2 for CHX10) occurring within the first 24 h. In mice, the CHX10 coating significantly reduced the number of CFU (colony forming units), both on the implants and in the peri-implant tissues, 1 d after S. aureus challenge. The CHX10-coated implants were well-tolerated by the animals, with no signs of toxicity observed by histological analysis. Moreover, the coating significantly reduced the frequency of culture-positive tissues 1 d, and of culture-positive implants 1 and 4 d after challenge. In summary, the chlorhexidine-releasing mechanically stable epoxy-based CHX10 coating prevented implant colonisation and S. aureus BAI in mice and has good prospects for clinical development.
Assuntos
Materiais Biocompatíveis/efeitos adversos , Clorexidina/uso terapêutico , Materiais Revestidos Biocompatíveis/química , Compostos de Epóxi/química , Próteses e Implantes/microbiologia , Infecções Relacionadas à Prótese/prevenção & controle , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Titânio/química , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Biópsia , Clorexidina/farmacologia , Liberação Controlada de Fármacos , Camundongos Endogâmicos C57BL , Infecções Relacionadas à Prótese/tratamento farmacológico , Infecções Relacionadas à Prótese/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologiaRESUMO
Biomaterial implants and devices increase the risk of microbial infections due to the biofilm mode of growth of infecting bacteria on implant materials, in which bacteria are protected against antibiotic treatment and the local immune system. Matrix-metalloproteinases (MMPs) and cell surface integrin receptors facilitate transmigration of inflammatory cells toward infected or inflamed tissue. This study investigates the relationship between MMP- and integrin-expression and the clearance of infecting Staphylococcus aureus around implanted biomaterials in a murine model.MMP- and integrin αvß3-expression were monitored in mice, with and without subcutaneously implanted biomaterial samples, in the absence and presence of bioluminescent S. aureus Xen36. Staphylococcal persistence was imaged longitudinally over time using bioluminescence imaging. The activatable MMPSense®680 and integrin-targeted IntegriSense®750 probes were injected on different days after implantation and their signal intensity and localisation monitored using fluorescence imaging. After sacrifice 7 or 16 days post-implantation, staphylococci from biomaterial samples and surrounding tissues were cultured on agar-plates and presence of host inflammatory cells was histologically evaluated.MMP- and integrin-expression were equally enhanced in presence of staphylococci or biomaterials up to 7 days post-implantation, but their localisation along the biomaterial samples differed. Bacterial clearance from tissue was higher in the absence of biomaterials. It is of clinical relevance that MMP- and integrin-expression were enhanced in presence of both staphylococci and biomaterials, although the immune system in the presence of biomaterials remained hampered in eradicating bacteria during the first 7 days post-implantation.
Assuntos
Biofilmes/crescimento & desenvolvimento , Implantes Experimentais/microbiologia , Integrina alfaVbeta3/metabolismo , Metaloproteinases da Matriz/metabolismo , Infecções Estafilocócicas/metabolismo , Animais , Integrina alfaVbeta3/genética , Metaloproteinases da Matriz/genética , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Infecções Estafilocócicas/patologiaRESUMO
BACKGROUND: The origin of surgical site and biomaterial-associated infection is still elusive. Micro-organisms contaminating the wound may come from the air in the operating theatre, the surgical team or the skin of the patient. The skin of patients is disinfected prior to surgery, but bacteria deeper in the skin (e.g. in sweat glands or sebaceous glands) may not be reached. METHODS: A preliminary cohort study was performed to study the origin of surgical site and biomaterial-associated infection between May 2020 and February 2021. In order to investigate whether cutaneous microbiota colonize the wound when released from the skin upon cutting, aerobic and anaerobic bacteria were isolated, quantified and identified from the skin of 99 patients undergoing trauma surgery, before and after skin disinfection, from knife blades and from the wound directly after the first cut. RESULTS: Ninety-nine percent of the patients were culture-positive before disinfection with chlorhexidine. Of these, 40% were still culture-positive after disinfection. Of these, 54% had a positive culture of the wound after cutting the skin. Twenty percent of the patients with a negative culture after disinfection had a positive wound culture after cutting the skin. Staphylococcus epidermidis and Cutibacterium acnes were the most commonly cultured bacterial species. In 9% of cases, more than 100 bacterial colonies were cultured from the wound; this may cause biomaterial-associated infection. CONCLUSION: Bacteria residing in the skin and not eradicated by disinfection may enter the surgical wound upon cutting, resulting in contamination which may cause biomaterial-associated infection.
Assuntos
Clorexidina , Infecção da Ferida Cirúrgica , Humanos , Estudos de Coortes , Infecção da Ferida Cirúrgica/microbiologia , Pele/microbiologia , Staphylococcus epidermidisRESUMO
Honey has potent activity against both antibiotic-sensitive and -resistant bacteria, and is an interesting agent for topical antimicrobial application to wounds. As honey is diluted by wound exudate, rapid bactericidal activity up to high dilution is a prerequisite for its successful application. We investigated the kinetics of the killing of antibiotic-resistant bacteria by RS honey, the source for the production of Revamil® medical-grade honey, and we aimed to enhance the rapid bactericidal activity of RS honey by enrichment with its endogenous compounds or the addition of antimicrobial peptides (AMPs). RS honey killed antibiotic-resistant isolates of Pseudomonas aeruginosa, Staphylococcus epidermidis, Enterococcus faecium, and Burkholderia cepacia within 2 h, but lacked such rapid activity against methicillin-resistant S. aureus (MRSA) and extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. It was not feasible to enhance the rapid activity of RS honey by enrichment with endogenous compounds, but RS honey enriched with 75 µM of the synthetic peptide Bactericidal Peptide 2 (BP2) showed rapid bactericidal activity against all species tested, including MRSA and ESBL E. coli, at up to 10-20-fold dilution. RS honey enriched with BP2 rapidly killed all bacteria tested and had a broader spectrum of bactericidal activity than either BP2 or honey alone.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Mel , Viabilidade Microbiana/efeitos dos fármacos , Bactérias/isolamento & purificação , HumanosRESUMO
Infection of biomedical devices is characterized by biofilm formation and colonization of surrounding tissue by the causative pathogens. To investigate whether bacteria detected microscopically in tissue surrounding infected devices were viable, we used bromodeoxyuridine (BrdU), a nucleotide analogue that is incorporated into bacterial DNA and can be detected with antibodies. Infected human tissue was obtained postmortem from patients with intravascular devices, and mouse biopsy specimens were obtained from mice with experimental biomaterial infection. In vitro experiments showed that Staphylococcus epidermidis incorporated BrdU, as judged from staining of the bacteria with anti-BrdU antibodies. After incubation of bacteria with BrdU and subsequent staining of microscopic sections with anti-BrdU antibodies, bacteria could be clearly visualized in the tissue surrounding intravascular devices of deceased patients. With this staining technique, relapse of infection could be visualized in mice challenged with a low dose of S. epidermidis and treated with dexamethasone between 14 and 21 days after challenge to suppress immunity. This confirms and extends our previous findings that pericatheter tissue is a reservoir for bacteria in biomaterial-associated infection. The pathogenesis of the infection and temporo-spatial distribution of viable, dividing bacteria can now be studied at the microscopic level by immunolabeling with BrdU and BrdU antibodies.
Assuntos
Técnicas Bacteriológicas/métodos , Bromodesoxiuridina/metabolismo , Infecções Relacionadas a Cateter/diagnóstico , Viabilidade Microbiana , Infecções Relacionadas à Prótese/diagnóstico , Infecções Estafilocócicas/diagnóstico , Staphylococcus epidermidis/isolamento & purificação , Animais , Materiais Biocompatíveis , Infecções Relacionadas a Cateter/microbiologia , Humanos , Imuno-Histoquímica/métodos , Camundongos , Microscopia/métodos , Infecções Relacionadas à Prótese/microbiologia , Coloração e Rotulagem/métodos , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/crescimento & desenvolvimento , Staphylococcus epidermidis/metabolismoRESUMO
BACKGROUND: Nowadays, personalized medical devices are frequently used for patients. Due to the manufacturing procedure sterilization is required. How different sterilization methods affect the mechanical behavior of these devices is largely unknown. MATERIALS AND METHODS: Three poly(methyl methacrylate) (PMMA) based materials (Vertex Self-Curing, Palacos R+G, and NextDent C&B MFH) were sterilized with different sterilization methods: ethylene oxide, hydrogen peroxide gas plasma, autoclavation, and γ-irradiation. Mechanical properties were determined by testing the flexural strength, flexural modulus, fracture toughness, and impact strength. RESULTS: The flexural strength of all materials was significantly higher after γ-irradiation compared to the control and other sterilization methods, as tested in a wet environment. NextDent C&B MFH showed the highest flexural and impact strength, Palacos R+G showed the highest maximum stress intensity factor and total fracture work. CONCLUSION: Autoclave sterilization is not suitable for the sterilization of PMMA-based materials. Ethylene oxide, hydrogen peroxide gas plasma, and γ-irradiation appear to be suitable techniques to sterilize PMMA-based personalized medical devices.
Assuntos
Fenômenos Mecânicos , Polimetil Metacrilato , Medicina de Precisão/instrumentação , Esterilização , Raios gama , Gases em Plasma/química , Polimetil Metacrilato/químicaRESUMO
The aim of this study was to cover the surfaces of zirconium (Zr) with an antimicrobial layer for biomedical applications. For this purpose, the micro-arc oxidation (MAO) process was employed in a sodium silicate and sodium hydroxide containing base electrolyte with and without addition of silver acetate (AgC2H3O2). In general, synthesized MAO layers were composed of zirconium oxide (ZrO2) and zircon (ZrSiO4). Addition of AgC2H3O2 into the base electrolyte caused homogenous precipitation of silver-containing particles in the MAO layer, which exhibited excellent antibacterial efficiency against methicillin-resistant Staphylococcus aureus (MRSA) as compared to the untreated and MAO-treated Zr.
Assuntos
Anti-Infecciosos , Materiais Revestidos Biocompatíveis , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Zircônio , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Silicatos/química , Prata/química , Hidróxido de Sódio/química , Zircônio/química , Zircônio/farmacologiaRESUMO
The scarcity of current antibiotic-based strategies to prevent biomaterial-associated infections (BAI) and their risk of resistance development prompted us to develop a novel antimicrobial implant-coating to prevent Staphylococcus aureus-induced BAI. We incorporated the antimicrobial peptide OP-145 into a Polymer-Lipid Encapsulation MatriX (PLEX)-coating to obtain high peptide levels for prolonged periods at the implant-tissue interphase. We first confirmed that OP-145 was highly effective in killing S. aureus and inhibiting biofilm formation in vitro. OP-145 injected along S. aureus-inoculated implants in mice significantly reduced the number of culture-positive implants. OP-145 was released from the PLEX coating in a controlled zero-order kinetic rate after an initial 55%-burst release and displayed bactericidal activity in vitro. In a rabbit intramedullary nail-related infection model, 67% of rabbits with PLEX-OP-145-coated nails had culture-negative nails after 28days compared to 29% of rabbits with uncoated nails. In rabbits with PLEX-OP-145-coated nails, bone and soft tissue samples were culture-negative in 67% and 80%, respectively, whereas all bone samples and 71% of the soft tissue samples of rabbits with uncoated nails were infected. Together, PLEX-OP-145 coatings, of which both compounds have already been found safe in man, can prevent implant colonization and S. aureus-induced BAIs.
Assuntos
Antibacterianos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Infecções Estafilocócicas/prevenção & controle , 1,2-Dipalmitoilfosfatidilcolina/química , Animais , Antibacterianos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Biofilmes , Colesterol/química , Feminino , Ácido Láctico/química , Camundongos Endogâmicos C57BL , Doenças da Unha/tratamento farmacológico , Fosfatidilcolinas/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Próteses e Implantes , Coelhos , Silicones/química , Staphylococcus aureus/efeitos dos fármacosRESUMO
Chemically cross-linked gelatin-chondroitin sulphate (ChS) hydrogels, impregnated in Dacron, were evaluated as drug delivery systems for antibacterial proteins. The gelatin-chondroitin sulphate gels, plain or impregnated in Dacron, were cross-linked with a water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The release of lysozyme and recombinant thrombocidin (rTC-1), an antibacterial protein derived from human blood platelets, from the gelatin-ChS gels in Dacron in phosphate-buffered saline at 37 degrees C was determined, and compared to the release from gelatin gels in Dacron and plain gelatin-ChS gels. The incorporation of chondroitin sulphate into gelatin gels, caused a marked increase in lysozyme loading capacity, and a slower release rate. The relative release profiles for rTC-1 and lysozyme were equal for cross-linked gelatin as well as for cross-linked gelatin-ChS gels. Furthermore, rTC-1 showed no loss of antibacterial activity after 1 week of release. The lysozyme concentration profiles in the samples and in the surrounding medium as a function of time were calculated using mathematical solutions for Ficks second law of diffusion for a semi-infinite composite medium, which is a schematic representation of a slab in a surrounding medium. The biocompatibility and degradation of the Dacron matrices impregnated with gelatin-ChS gels was studied after implantation in subcutaneous pockets in rats. Chemically cross-linked gelatin-Ch5 gels showed a mild tissue reaction, and almost complete degradation within 18 weeks of implantation.
Assuntos
Anti-Infecciosos/administração & dosagem , Sulfatos de Condroitina/administração & dosagem , Gelatina/administração & dosagem , Hidrogéis/administração & dosagem , Proteínas/administração & dosagem , Animais , Materiais Biocompatíveis , Humanos , Técnicas In Vitro , Masculino , Muramidase/administração & dosagem , Ratos , Ratos WistarRESUMO
We demonstrate that the 1C10 monoclonal antibody (mAb) directed against the N-terminal domain of the colicin A recognizes a 13 residue-region (13Thr-Gly-Trp-Ser-Ser-Glu-Arg-Gly-Ser-Gly-Pro- Asp-Pro25). When this peptide is inserted into a protein in the amino-terminal or an internal position, the tagged protein is efficiently detected by the 1C11 mAb either by immunoblotting or immunoprecipitation. In vitro, the minimal structure required for detection using the pepscan system is 19Arg-Gly-Ser-Gly-Pro-Glu-Pro25, indicating that in vivo the proper exposure of the epitope requires additional residues. The construction of a versatile vector allowing overproduction of tagged proteins is described. Various applications of the 1C11 epitope are mentioned. This epitope did not alter the function of any of the proteins so far tested.
Assuntos
Colicinas/imunologia , Epitopos/imunologia , Escherichia coli/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Sequência de Bases , Clonagem Molecular , Colicinas/genética , DNA Bacteriano , Escherichia coli/genética , Vetores Genéticos , Immunoblotting , Dados de Sequência MolecularRESUMO
To determine the effect of Pseudomonas aeruginosa exotoxin A (P-ExA) on cytokine production, we studied cytokine release induced by heat-killed P. aeruginosa (HKPA) in human whole blood in the presence or absence of P-ExA. P-ExA (0.01-1 microgram ml(-1)) caused a dose-dependent decrease in HKPA-induced production of tumor necrosis factor alpha (TNF), interleukin (IL-) 10, IL-6 and IL-8 (all P<0.05). P-ExA-induced inhibition of IL-10, IL-6 and IL-8 release was not dependent on reduced TNF concentrations, since the relative attenuation of the production of these cytokines was similar in the presence or absence of a neutralizing anti-TNF antibody. The effect of P-ExA on cytokine production may offer a disadvantage to the host with respect to clearance of the infection.
Assuntos
ADP Ribose Transferases , Toxinas Bacterianas/imunologia , Citocinas/sangue , Exotoxinas/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/patogenicidade , Fatores de Virulência , Toxinas Bacterianas/farmacologia , Sobrevivência Celular , Citocinas/biossíntese , Exotoxinas/farmacologia , Temperatura Alta , Humanos , Técnicas In Vitro , Interleucina-10/biossíntese , Interleucina-10/sangue , Interleucina-6/biossíntese , Interleucina-6/sangue , Interleucina-8/biossíntese , Interleucina-8/sangue , Infecções por Pseudomonas/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Virulência , Exotoxina A de Pseudomonas aeruginosaRESUMO
Prosthetic valve endocarditis may be reduced by the local delivery of antibacterial proteins from the Dacron sewing ring of a prosthetic heart valve. Dacron discs were treated with a carbon dioxide gas plasma to improve the hydrophilicity and thereby enabling homogeneous impregnation with gelatin type B. The gelatin samples were cross-linked to different degrees using various amounts of water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Lysozyme, a model protein for antibacterial proteins, was loaded into (non)-cross-linked gelatin gels incorporated in Dacron, or adsorbed onto non-treated and gas plasma-treated Dacron. The in vivo lysozyme release was measured after subcutaneous implantation of lysozyme-loaded samples in rats. The lysozyme content of the samples, and the lysozyme level of the surrounding tissue were determined at different explantation times (ranging from 6 h up to 1 week). For cross-linked gelatin gels, the lysozyme tissue level was elevated up to 2 days after implantation. In vitro release was measured using agarose medium or phosphate buffer. Lysozyme release in buffer solution under sink conditions was in good agreement with the in vivo lysozyme release profiles, and therefore considered a good model to describe in vivo release characteristics. The release was modelled with a solution of Fick's second law of diffusion using the appropriate boundary conditions. In this way the lysozyme concentration in the gel and the surrounding tissue as a function of time and distance was obtained. The presence of cross-linked gelatin in Dacron did lead to an increased uptake of lysozyme and a delayed release during 30 h after implantation, whereas a burst release took place from Dacron, gas plasma-treated Dacron, or Dacron containing non-cross-linked gelatin.
Assuntos
Anti-Infecciosos/administração & dosagem , Excipientes/química , Gelatina/química , Próteses Valvulares Cardíacas , Hidrogéis/química , Muramidase/administração & dosagem , Algoritmos , Animais , Anti-Infecciosos/química , Carbodi-Imidas/química , Dióxido de Carbono/química , Reagentes de Ligações Cruzadas , Difusão , Indicadores e Reagentes , Modelos Teóricos , Muramidase/química , Polietilenotereftalatos/química , Ratos , EsterilizaçãoRESUMO
Prosthetic valve endocarditis is an infrequent, but serious complication of cardiac valve replacement. The infection is caused by the adherence of bacteria to the prosthetic valve or to tissue at the site of implantation. Recently it was shown that antibacterial peptides from blood platelets are involved in clearance and killing of bacteria adhering to vegetations induced in a model for prosthetic valve endocarditis using rabbits. The application of these antibacterial proteins in a release system, incorporated in the Dacron sewing ring of the prosthetic heart valve would diminish the incidence of endocarditis. In this study a release system for small cationic proteins based on cross-linked gelatin was developed and characterised. Furthermore, the system was evaluated with respect to the uptake and in vitro release of lysozyme, a small cationic protein that was chosen as a model protein for small cationic antibacterial proteins. Variation of gelatin type (A and B), and cross-link density resulted in differences in swelling, thermal behaviour, and number of charged groups. Lysozyme uptake was proportional to swelling, but was governed by the number of anionic groups. The latter was also observed for the release profiles: when the amount of free carboxylic acids is higher (gelatin B compared to gelatin A), the lysozyme release lasts for a longer time period. The release into solidified agarose medium, as a model for heart muscle tissue, was measured. After 50 h, 40-100% of the lysozyme was released, which is in accordance with the aimed release period of 24-48 h. The adsorption experiments in vitro suggest an influence of the electrostatic interactions between lysozyme and gelatin. This hypothesis was validated with a mathematical model which takes both diffusion and adsorption interactions into account.
Assuntos
Antibacterianos/administração & dosagem , Materiais Biocompatíveis , Proteínas/administração & dosagem , Animais , Varredura Diferencial de Calorimetria , Gelatina/metabolismo , Modelos Químicos , Muramidase/metabolismo , CoelhosRESUMO
Implant-associated infections (IAIs) may be prevented by providing antibacterial properties to the implant surface prior to implantation. Using a plasma electrolytic oxidation (PEO) technique, we produced porous TiO2 coatings bearing various concentrations of Ag nanoparticles (Ag NPs) (designated as 0 Ag, 0.3 Ag and 3.0 Ag) on a Ti-6Al-7Nb biomedical alloy. This study investigates the cytotoxicity of these coatings using a human osteoblastic cell line (SV-HFO) and evaluates their bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA). The release of Ag and the total amount of Ag in the coatings were determined using a graphite furnace atomic absorption spectrometry technique (GF-AAS) and flame-AAS, respectively. Cytotoxicity was evaluated using the AlamarBlue assay coupled with the scanning electron microscopy (SEM) observation of seeded cells and by fluorescence microscopy examination of the actin cytoskeleton and nuclei after 48 h of incubation. Antibacterial activity was assessed quantitatively using a direct contact assay. AlamarBlue viability assay, SEM and fluorescence microscopy observation of the SV-HFO cells showed no toxicity for 0 Ag and 0.3 Ag specimens, after 2, 5 and 7 days of culture, while 3.0 Ag surfaces appeared to be extremely cytotoxic. All Ag-bearing surfaces had good antibacterial activity, whereas Ag-free coatings showed an increase in bacterial numbers. Our results show that the 0.3 Ag coatings offer conditions for optimum cell growth next to antibacterial properties, which makes them extremely useful for the development of new antibacterial dental and orthopedic implants.
Assuntos
Antibacterianos/farmacologia , Materiais Revestidos Biocompatíveis/farmacologia , Feto/citologia , Osteoblastos/citologia , Prata/farmacologia , Titânio/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Transformada , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Eletrólitos/química , Humanos , Viabilidade Microbiana/efeitos dos fármacos , Microscopia de Fluorescência , Osteoblastos/efeitos dos fármacos , Osteoblastos/ultraestrutura , Oxirredução/efeitos dos fármacos , PorosidadeRESUMO
Honey has been used successfully in wound healing for thousands of years. The peptide hormone human epidermal growth factor (hEGF) is also known to have a beneficial effect in various wound healing processes via mechanisms that differ from those for honey. In this study, we show that hEGF can be incorporated into honey via nectar. Plants of Nicotiana langsdorffii x N. sanderae were transformed with the gene for hEGF, equipped with a nectary-targeted promoter and a signal sequence for secretion to nectar. These plants accumulated hEGF in the nectar. The maximum hEGF concentration recorded with ELISA in these plants is 2.5 ng·ml⻹. There is a significant linear relationship (P<0.001) between hEGF concentration and induction of hEGF-receptor phosphorylation. Since the flower morphology of these plants did not allow production of honey from their nectar, we used feeding solutions, spiked with synthetic hEGF, to study transfer of this peptide into honey through bee activity. Transfer of hEGF from a feeding solution to honey by bees occurred with retention of the hEGF concentration and the capacity to induce hEGF-receptor phosphorylation. These observations indicate that plants can function as a production platform for honey containing biologically active peptides, which may enhance wound healing and other biological processes.
Assuntos
Abelhas , Fator de Crescimento Epidérmico/biossíntese , Fator de Crescimento Epidérmico/genética , Mel , Nicotiana/genética , Nicotiana/metabolismo , Néctar de Plantas/metabolismo , Animais , Flores/genética , Flores/metabolismo , Humanos , Plantas Geneticamente Modificadas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Cicatrização/efeitos dos fármacosAssuntos
Endocardite Bacteriana/etiologia , Genes Bacterianos , Regiões Promotoras Genéticas , Streptococcus/genética , Streptococcus/patogenicidade , Atividade Bactericida do Sangue , Plaquetas/fisiologia , Endocardite Bacteriana/microbiologia , Fibrina/fisiologia , Expressão Gênica , Vetores Genéticos , Humanos , Técnicas In Vitro , Modelos Biológicos , Streptococcus sanguis/genética , Streptococcus sanguis/patogenicidade , Virulência/genéticaAssuntos
Aderência Bacteriana/efeitos dos fármacos , Atividade Bactericida do Sangue , Plaquetas/fisiologia , Glucanos/farmacologia , Streptococcus/efeitos dos fármacos , Streptococcus/patogenicidade , Aderência Bacteriana/fisiologia , Proteínas Sanguíneas/isolamento & purificação , Proteínas Sanguíneas/farmacologia , Endocardite Bacteriana/etiologia , Humanos , Técnicas In Vitro , Infecções Estreptocócicas/etiologia , Streptococcus/fisiologiaRESUMO
Isolation of plasmid DNA from viridans group streptococci is difficult, and preparations are often heavily contaminated with chromosomal DNA. We developed a simple protocol to isolate pure plasmid DNA for use in different molecular techniques, including automated sequencing. The protocol is also applicable for plasmid isolation from Staphylococcus aureus. In addition, the protocol allows isolation of pure endogenous plasmids from streptococci and S. aureus.
Assuntos
Plasmídeos , Staphylococcus aureus/genética , Streptococcus/genética , Clonagem Molecular , Vetores GenéticosRESUMO
Viridans group streptococci (VS) from the oral cavity entering the bloodstream may initiate infective endocarditis (IE). We aimed to identify genes expressed in response to a pH increase from slightly acidic (pH 6.2) to neutral (pH 7.3) as encountered by VS entering the bloodstream from the oral cavity. Using a recently developed promoter-screening vector, we isolated five promoter fragments from the genomic DNA of Streptococcus gordonii CH1 responding to this stimulus. No common regulatory sequences were identified in these promoter fragments that could account for the coordinate expression of the corresponding genes. One of the isolated fragments contained the promoter region and 5' end of a gene highly homologous to the methionine sulfoxide reductase gene (msrA) of various bacterial and eukaryotic species. This gene has been found to be activated in S. gordonii strain V288 in a rabbit model of IE (A. O. Kiliç, M. C. Herzberg, M. W. Meyer, X. Zhao, and L. Tao, Plasmid 42:67-72, 1999). We isolated and characterized the msrA gene of S. gordonii CH1 and constructed a chromosomal insertion mutant. This mutant was more sensitive to hydrogen peroxide, suggesting a role for the streptococcal MsrA in protecting against oxidative stress. Moreover, MsrA appeared to be important for the growth of S. gordonii CH1 under aerobic and anaerobic conditions. Both these properties of MsrA may contribute to the ability of S. gordonii to cause IE.