OCIMUM SANCTUM EXTRACT COATING ON BIOMATERIAL SURFACES TO PREVENT BACTERIAL ADHESION AND BIOFILM GROWTH

Objective: The objective of this work is to evaluate the performance of OS extract as a coating on biomaterial surfaces in preventing bacterial adhesionand biofilm growth, as an effective measure to combat Biomaterial associated infections.Methods: Here, we have incorporated the extract from a medicinal plant as a coating to biomaterial surfaces in order to prevent bacterial adhesionand biofilm growth. To this end, Ocimum sanctum (OS) oil extract is coated on biomaterials (polymethyl methacrylate and polystyrene) and bacteriasuch as Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were allowed to adhere and grow for 1 hr, 3 hrs and 24 hrs.Results: A significant reduction (p<0.01) in number of adherent bacteria on OS extract coated surfaces compared to bare surfaces was observed atall-time points. The zone of inhibition of OS extract was observed for all the three bacteria and maximum inhibition was observed for P. aeruginosa(30 mm diameter) compared to S. aureus (25 mm diameter) and E. coli (28 mm diameter).Conclusion: Thus, OS oil extract could be a promising coating for reduction of bacterial adhesion and biofilm formation.Keywords: Antibacterial coating, Bacterial adhesion, Biofilm, Biomaterial, Biomaterials-associated infection, Ocimum sanctum

Antibacterial Efficacy of Iron-Oxide Nanoparticles against Biofilms on Different Biomaterial Surfaces

Biofilm growth on the implant surface is the number one cause of the failure of the implants. Biofilms on implant surfaces are hard to eliminate by antibiotics due to the protection offered by the exopolymeric substances that embed the organisms in a matrix, impenetrable for most antibiotics and immune cells. Application of metals in nanoscale is considered to resolve biofilm formation. Here we studied the effect of iron-oxide nanoparticles over biofilm formation on different biomaterial surfaces and pluronic coated surfaces. Bacterial adhesion for 30 min showed significant reduction in bacterial adhesion on pluronic coated surfaces compared to other surfaces. Subsequently, bacteria were allowed to grow for 24 h in the presence of different concentrations of iron-oxide nanoparticles. A significant reduction in biofilm growth was observed in the presence of the highest concentration of iron-oxide nanoparticles on pluronic coated surfaces compared to other surfaces. Therefore, combination of polymer brush coating and iron-oxide nanoparticles could show a significant reduction in biofilm formation

Microbial biofilm growth vs. tissue integration: "the race for the surface" experimentally studied

Biomaterial-associated infections constitute a major clinical problem. Unfortunately, microorganisms are frequently introduced onto an implant surface during surgery and start the race for the surface before tissue integration can occur. So far, no method has been forwarded to study biofilm formation and tissue integration simultaneously. The aim of this study is to describe an in vitro method to investigate this “race for the surface”. First, a suitable growth medium was prepared that allowed both bacterial and tissue growth in a parallel plate flow chamber. Staphylococci were deposited on the glass bottom plate of the flow chamber in different surface densities, after which U2OS osteosarcoma cells were seeded. U2OS cells did not grow in the absence of flow, possibly due to poisoning by bacterial endotoxins, but under flow both staphylococci and U2OS cells grew. The number of adhering cells and area per spread cell were determined after 48 h in relation to the initial number of bacteria present. Both the number and spread area per cell decreased with increasing density of adhering staphylococci. This demonstrates that the outcome of the race for the surface between bacteria and tissue cells is dependent on the number of bacteria present prior to cell seeding.\u

In Vitro Interactions between Bacteria, Osteoblast-Like Cells and Macrophages in the Pathogenesis of Biomaterial-Associated Infections

Biomaterial-associated infections constitute a major clinical problem that is difficult to treat and often necessitates implant replacement. Pathogens can be introduced on an implant surface during surgery and compete with host cells attempting to integrate the implant. The fate of a biomaterial implant depends on the outcome of this race for the surface. Here we studied the competition between different bacterial strains and human U2OS osteoblast-like cells (ATCC HTB-94) for a poly(methylmethacrylate) surface in the absence or presence of macrophages in vitro using a peri-operative contamination model. Bacteria were seeded on the surface at a shear rate of 11 1/s prior to adhesion of U2OS cells and macrophages. Next, bacteria, U2OS cells and macrophages were allowed to grow simultaneously under low shear conditions (0.14 1/s). The outcome of the competition between bacteria and U2OS cells for the surface critically depended on bacterial virulence. In absence of macrophages, highly virulent Staphylococcus aureus or Pseudomonas aeruginosa stimulated U2OS cell death within 18 h of simultaneous growth on a surface. Moreover, these strains also caused cell death despite phagocytosis of adhering bacteria in presence of murine macrophages. Thus U2OS cells are bound to loose the race for a biomaterial surface against S. aureus or P. aeruginosa, even in presence of macrophages. In contrast, low-virulent Staphylococcus epidermidis did not cause U2OS cell death even after 48 h, regardless of the absence or presence of macrophages. Clinically, S. aureus and P. aeruginosa are known to yield acute and severe biomaterial-associated infections in contrast to S. epidermidis, mostly known to cause more low-grade infection. Thus it can be concluded that the model described possesses features concurring with clinical observations and therewith has potential for further studies on the simultaneous competition for an implant surface between tissue cells and pathogenic bacteria in presence of immune system components

Bridging the gap between in vitro and in vivo evaluation of biomaterials-associated infections

Hoe ouder we met zijn allen worden, hoe vaker we gebruik zullen maken van implantaten als kunstheupen, kunstknieën en pacemakers. Gevaar bij dergelijke implantaten is dat zich micro-organismen (bijvoorbeeld bacteriën) aan het oppervlak kunnen vasthechten. Er ontstaat dan een biofilm, vergelijkbaar met roest en tandplak, die onder meer infecties kan veroorzaken en het implantaat kan beschadigen. Promovendus Guruprakash Subbiahdoss bracht in kaart hoe groot de kans is dat een biofilm ontstaat op een implantaat. Zelfs als er maar een heel klein aantal bacteriën aanwezig is, kan er een biofilm ontstaan, zo blijkt. Dit hangt af van de virulentiefactor van de ziektekiem. Het is dan ook nodig om implantaten en coatings voor implantaten te ontwikkelen, die de groei van bacteriën tegengaan, en de groei van gezond weefsel rondom het implantaat stimuleren. Het huidige onderzoek hiernaar kan worden verbeterd door aan laboratoriumstudies met weefselkweken ook antibiotica, groeifactoren en cytokines toe te voegen, stelt Subbiahdoss. Zo wordt de in vivo situatie beter gesimuleerd. Hij stelt voor om de resultaten uit onderzoek met weefselkweken te toetsen in diermodellen. Door de materialen en coatings goed te screenen, kan het aantal benodigde proefdieren beperkt blijven. Biomaterials play an important role in modern medicine in the restoration of tissue, organ or body function. The use of biomaterial implants and medical devices is mainly restricted by complications due to biomaterial-associated infections (BAI). There are various routes along which microorganisms can enter the body and develop a BAI in the case of permanent implants. The best-documented route is direct contamination of an implant during surgery (peri-operative contamination). BAI can also be initiated immediately post-surgery during hospitalization (post-operative contamination) or microbial spreading through blood from infections elsewhere in the human body. In 1987, the orthopedic surgeon Anthony Gristina coined the term “race for the surface” that is the fate of a biomaterial implant was depicted as a race between microbial adhesion and biofilm growth on an implant surface versus tissue integration. Irrespective of the route of infection, the fate of biomaterial implants depends mainly on the outcome of the so-called ‘race for the surface’. Till today, biomaterials or functional coatings were evaluated in vitro either for their ability to resist bacterial adhesion or for their ability to support mammalian cell adhesion and proliferation based on the concept of the race for the surface. However, no attempt was made to address the simultaneous effects of the presence of bacteria and mammalian cells on a biomaterial surface due to the lack of proper methodology, which according to the concept of the ‘race for the biomaterial surface’, is crucial for the ultimate fate of a biomaterial implant. A proper method to study the race for the surface on an experimental basis, would constitute a valuable bridge between in vivo and in vitro studies. Therefore, this thesis focuses on the development of methods that could bridge the gap between in vitro and in vivo studies on BAI.

Mammalian cell growth versus biofilm formation on biomaterial surfaces in an in vitro post-operative contamination model

Biomaterial-associated infections are the major cause of implant failure and can develop many years after implantation. Success or failure of an implant depends on the balance between host tissue integration and bacterial colonization. Here, we describe a new in vitro model for the postoperative bacterial contamination of implant surfaces and investigate the effects of contamination on the balance between mammalian cell growth and bacterial biofilm formation. U2OS osteosarcoma cells were seeded on poly(methyl methacrylate) in different densities and allowed to grow for 24 h in a parallel-plate flow chamber at a low shear rate (0.14 s(-1)), followed by contamination with Staphylococcus epidermidis ATCC 35983 at a shear rate of 11 s(-1). The U2OS cells and staphylococci were allowed to grow simultaneously for another 24 h under low-shear conditions (0.14 s(-1)). Mammalian cell growth was severely impaired when the bacteria were introduced to surfaces with a low initial cell density (2.5x10(4) cells cm(-2)), but in the presence of higher initial cell densities (8.2x10(4) cells cm(-2) and 17x10(4) cells cm(-2)), contaminating staphylococci did not affect cell growth. This study is believed to be the first to show that a critical coverage by mammalian cells is needed to effectively protect a biomaterial implant against contaminating bacteria