Methodologies for <i>in vitro</i> and <i>in vivo</i> evaluation of efficacy of antifungal and antibiofilm agents and surface coatings against fungal biofilms.

Unlike superficial fungal infections of the skin and nails, which are the most common fungal diseases in humans, invasive fungal infections carry high morbidity and mortality, particularly those associated with biofilm formation on indwelling medical devices. Therapeutic management of these complex diseases is often complicated by the rise in resistance to the commonly used antifungal agents. Therefore, the availability of accurate susceptibility testing methods for determining antifungal resistance, as well as discovery of novel antifungal and antibiofilm agents, are key priorities in medical mycology research. To direct advancements in this field, here we present an overview of the methods currently available for determining (i) the susceptibility or resistance of fungal isolates or biofilms to antifungal or antibiofilm compounds and compound combinations; (ii) the &lt;i&gt;in vivo&lt;/i&gt; efficacy of antifungal and antibiofilm compounds and compound combinations; and (iii) the &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt; performance of anti-infective coatings and materials to prevent fungal biofilm-based infections

INVOLVEMENT OF BACTERICIDAL FACTORS FROM THROMBIN-STIMULATED PLATELETS IN CLEARANCE OF ADHERENT VIRIDANS STREPTOCOCCI IN EXPERIMENTAL INFECTIVE ENDOCARDITIS

Platelets activated with thrombin release bactericidal factors. We studied the role of the susceptibility of viridans streptococci to these bactericidal factors in the development of infective endocarditis (IE). By using the experimental endocarditis rabbit model, the initial adherence and the development of IE, were assessed for 10 viridans streptococcal strains differing in their susceptibilities to releasate (material released) from thrombin-activated platelets. Six strains were susceptible and four strains were resistant to these releasates. The numbers of vegetations (VGs) colonized at 5 min and 48 h after intravenous challenge with 10(4) CPU were determined. At 5 min after challenge, significantly more VGs were colonized with bacteria of the six platelet releasate-susceptible strains than with bacteria of the four releasate-resistant strains (P <0.005). In the reIeasate-susceptible group of strains, the number of colonized VGs decreased significantly between 5 min and 48 h after intravenous inoculation (P <0.001). Such a decrease was not observed with the releasate-resistant strains. As a result, the final developments of IE due to releasate-susceptible and -resistant strains were not significantly different. The releasate-susceptible strain I and the releasate-resistant strain 2 were selected for more detailed experiments. Rabbits were killed at 5 and 30 min and 2, 4, and 48 h after inoculation. The number of culture-positive VGs as well as the number of adherent bacteria on the individual VGs were determined. The 90% infective dose for each strain was 10(5) CFU. At low inoculum concentrations (10(3) and 10(4) CFU) a larger proportion of the inoculated bacteria of both strains was found to be adherent on VGs than at higher challenge doses. The number of culture-positive VGs as well as the number of adherent bacteria per VG decreased rapidly in the first 30 min after challenge with strain 1 but not after challenge with strain 2. Additional experiments with the platelet releasate-susceptible strain S224 and the platelet releasate-resistant strain S182 confirmed the data obtained with strains 1 and 2 and indicated that releasate-susceptible strains disappeared from the VGs with time, whereas releasate-resistant strains persisted. In vitro studies with VGs excised 5 min after challenge with strain 1 or 2 showed that clearance of the releasate-susceptible strain 1 was not caused by complement bactericidal activity or surface phagocytosis by polymorphonuclear cells. Bacterial cells of strain I adherent on excised VGs were rapidly cleared by exposure to fresh clotting blood or to releasates from thrombin-stimulated platelet suspensions. In contrast, strain 2 bacteria adherent on VGs were hardly affected by these treatments. These data strongly indicate that bactericidal factors released from platelets upon thrombin stimulation are involved in the clearance of bacteria early after their adherence to VGs. Therefore, development of IE is the combined result of the abilities of viridans streptococci to adhere to VGs and to resist the activity of platelet-released bactericidal factors

Presence of a biomaterial implant facilitates induction of experimental infective endocarditis due to streptococci and staphylococci

Infective endocarditis (IE) usually is studied using animals with catheters inserted into the heart, which causes formation of platelet-fibrin thrombi (vegetations, VGs). We used two rabbit models to study the respective roles of the catheter and the VGs in the development of IE. The influence of the catheter was studied by either removing the catheter before bacterial challenge, or leaving the catheter in place. In all cases, removal of the catheter caused a strong decrease in the frequency of IE. The presence of the catheter stimulated population increase of streptococci within 4 h after challenge. As most catheters were sterile 4 h after challenge, they did not serve as a reservoir of bacteria. To study the requirement of a preformed VG catheters were inserted either 24 h or 30 min before bacterial challenge. In the former model VGs were present, in the latter VGs were not yet formed when bacteria were injected. The frequencies of IE due to 2 S. sanguis and 2 S. epidermidis strains in the 24 h model or 30 min model were similar, indicating that a preformed VG is not necessary for development of IE. Five coagulase-negative stains were shown to vary in their capacity to cause IE in the 30 min model. Variation was not caused by differences in early adhesion br colonization of the aortic valve, but reflects differences in persistence after initial colonization. Like in the 24 h model, persistence of the bacteria was greatly enhanced by the continuous presence of the catheter. Possible mechanisms of the infection-potentiating effect of the catheters are discussed.</p

In vitro cytotoxicity evaluation of porous TiO2-Ag antibacterial coatings for human fetal osteoblasts

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 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. (C) 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved

Real-time quantification of matrix metalloproteinase and integrin αvβ3 expression during biomaterial-associated infection in a murine model

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 αvβ3-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

Genetically Modified Lactococcus lactis for Delivery of Human Interleukin-10 to Dendritic Cells

Interleukin-10 (IL-10) plays an indispensable role in mucosal tolerance by programming dendritic cells (DCs) to induce suppressor Th-cells. We have tested the modulating effect of L. lactis secreting human IL-10 (L. lactis(IL-10)) on DC function in vitro. Monocyte-derived DC incubated with L. lactis(IL-10) induced effector Th-cells thatmarkedly suppressed the proliferation of allogenic Th-cells as compared to L. lactis. This suppressive effect was only seen when DC showed increased CD83 and CD86 expression. Furthermore, enhanced production of IL-10 was measured in both L. lactis(IL-10)-derived DC and Th-cells compared to L. lactis-derived DC and Th-cells. Neutralizing IL-10 duringDC-Th-cell interaction and coculturing L. lactis(IL-10)-derived suppressor Th-cells with allogenic Th-cells in a transwell system prevented the induction of suppressor Th-cells. Only 130 pg/mL of bacterial-derived IL-10 and 40 times more exogenously added recombinant human IL-10 were needed during DC priming for the generation of suppressor Th-cells. The spatially restricted delivery of IL-10 by food-grade bacteria is a promising strategy to induce suppressor Th-cells in vivo and to treat inflammatory diseases