Periodic Report Summary 2 - EMBEK1 (Development and analysis of polymer based multi-functional bactericidal materials)
Project context and objectives:
The central objectives of the EMBEK1 programme were to gain an improved understanding how bacteria interact and colonise surfaces, how new polymers can be made to prevent bacterial attachment and growth, and how current technologies can be used to obtain real-time, high resolution information on the nano-scale mechanisms of bacteria - surface interactions.
The project aimed to study how changes in a bacterium's genome (mutant libraries) affect the phenotypic behaviour of bacteria in terms of:
(1) their virulence;
(2) their ability to attach and colonise modified surfaces and finally,
(3) their ability to infect and kill whole organisms (insects being used as a model system for studying bacterial infection).
In order to achieve this, nano-composite and nano-structured polymeric films were to be developed using specially designed precursor molecules to create surfaces which will both directly affect a bacterium's ability to attach and will also supply antimicrobial components to kill those bacteria that do succeed in attaching to the modified surface(s). Since some of the surfaces were to be developed for clinical applications, mammalian cytotoxicity and antibacterial persistence were key issues to be studied.
Novel nano-scale material science and physical chemistry techniques were used to create and understand antimicrobial surfaces. Sophisticated molecular microbiology techniques were used to understand the genetic components governing the interaction of a bacterial biotic cell surface with the novel antimicrobial surfaces. Furthermore, the phenotypic differences of bacteria exposed to the novel antimicrobial materials were investigated by comparative proteome analysis. This duel systems approach allowed us to theoretically model the processes of bacterial attachment and survival, which in turn enabled us to improve these surfaces in an iterative approach.
Project results:
In accordance with the goals set out in Annex 1, EMBEK1 created foreground in four main areas:
(A) Materials: The development of materials and in depth analysis of properties with feedback from biological studies and modelling
Foreground: Improved knowledge in surface engineering
A catalogue of new materials including metal ion release systems with controllable release properties showing different levels of sensitivity, stability, functionality and longevity. Alternative materials that include antimicrobial nano-particles embedded in polymer thin films, bio-responsive vesicles and surface immobilised bacteriophages that respond to pathogenic bacteria only. Several publications, follow-up project (FP7-2009, Grant Agreement No. 245500)
(B) Methods: The optimisation of current analytical technologies and development of new technologies to obtain real-time, high resolution information on the nano-scale mechanisms of bacteria
Foreground: Improved knowledge on design of experiments and techniques
Establishing standard operating procedures for antimicrobial- and cytotoxicity testing. New impedimetrically based sensor to sense pathogenic bacteria. New advanced protocol vor simultaneous microevolution of resistances and determination of MIC. Nano-LC/MALDI for total proteome analysis. Theoretical and biological modelling on different levels that feed back into materials development. Several publications in print, others in preparation.
(C) Biology: To improve our understanding of how bacteria colonise surfaces using sophisticated theoretical and biological modelling and optimise current analytical technologies to obtain real-time, high resolution information on the nano-scale mechanisms of bacteria
Foreground: Improved knowledge with results indicating:
- that the pathogenic activity of P. asymbiotica in an insect relates to that in a mammal;
- that the evolution of silver ion resistance in P. aeruginosa and S. aureus is highly unlikely - as shown by the inability to develop mutant libraries over the course of the project;
- that evolution of copper Schiff's Base resistance in S.aureus and P. aeruginosa is possible and first insights into mechanisms have been achieved;
- that proteins and small molecules (such as Glutathione) in normal bodily fluids are able to protect the bacteria by binding to free silver, copper and zinc in solution, thus possibly reducing the antimicrobial effect of the metal;
- the impact of mutations in attachment and biofilm formation in insect model infections.
(D) Feasibility: Demonstrators and prototypes exploring the feasibility of the solutions
Foreground: Methods for coating different 3-dimensional (3D) implants and textile materials. New collaborations, first in-vivo tests planned for selected materials, ethics applications in progress, projects in planning, patents, potential for licensing.
As such the project EMBEK1 has fulfilled all of the goals proposed in Annex 1.
Work during the project was carried out by a total of 46 researchers over the time of 3 years. We have published 12 papers in peer reviewed journals and anticipate at least 6 others to be submitted in the near future. Three patents were filed from efforts out of the EMBEK1 project and a follow up project (BACTERIOSAFE, Grant Agreement No. 245500) was started in July 2010 with science and technology (S&T) development specifically for wound management. Smaller collaborative projects have been initiated with experts at other institutions and companies.
Potential impact:
EMBEK1 succeeded in developing a catalogue of new materials based on nano-composite, metal release systems that could be tailored in their antimicrobial efficacy, longevity of properties, stability and cytocompatibility. EMBEK1 explored the currently developing area of using nanosized and microsized biocompatible containers in the form of vesicles, which can be loaded with a desired drug or antimicrobial agent and release this upon a biological stimulus. The programme has created a large number of materials that show promising results for applications mostly as short term or single-use items. Examples of where this would be of relevance includes a number of implant devices where infection is a threat during the first 10 - 14 days of implantation, a variety of hygiene articles, diagnostic kits and one-way articles of medical clothing. In this context selected coatings were already transferred to a pre-pilot scale plant and first textile materials have been tested.
EMBEK1 developed not only antimicrobial materials and coatings, but also coatings with multiple functionalities, where antimicrobial activity is combined with cell adhesion and proliferation; or antimicrobial activity with anti-adhesive properties without cytotoxicity. Implants and devices coated with such materials offer the unique and currently not commercially available possibility to ensure reduced infection with optimum bio-integration of the implant. This work has led to a patent application by the EMBEK1 partner and will be subject of further studies and exploitation in the future.
Biological studies with the nano-composite metal release systems developed in EMBEK1 revealed highly relevant data that may influence the use and further development of silver, zinc, copper as well as phage containing wound bandages, devices, creams and the like. The work has shown, for example, that at relatively low release dose into a wound (or the environment) silver is more effective towards P. aeruginosa, while zinc is more effective towards S. aureus. At higher dosage silver is effective towards all bacterial strains tested during the work, which supports results from the literature and is in line with the currently frequent use of silver as an antimicrobial agent in wound healing. The higher dosage of silver is however cause for concern, because of issues of toxicity and reduced cell differentiation in wound healing as recently reported in the literature.
EMBEK1 investigated the response of the whole organism (insect model) towards silver and selected silver containing materials developed in EMBEK1. Sophisticated methods of proteomics and gene sequencing were used to study the effect of silver, zinc and copper release systems towards different bacteria (Photorhabdus asymbiotica, Staphylococcus aureus and Pseudomonas aeruginosa) with the aim to understand mechanisms of resistance. In this context, the complete genome of Photorhabdus asymbiotica was decoded and has been made available to academia worldwide for future studies via the UNEXE (as well as the EMBEK1) website. Tests suggest that the sequencing results for Photorhabdus asymbiotica is equally relevant for studies on S. aureus and P. aeruginosa and will be of tremendous importance in upcoming follow up projects.
In several independent studies by the different groups in EMBEK1, it was shown that S. aureus and P. aeruginosa seems to be unable to evolve resistance towards silver. Over a period of 18 months EMBEK1 scientist could not evolve mutant libraries using silver, or silver containing materials. This result is in good agreement with the literature where resistances against silver coatings is barely described and is of tremendous importance to society, to product development and industrial processing, in particular in light of the rapid spread of MRSA and the ability of bacteria, such as P. aeruginosa and S. aureus, to develop resistance towards antibiotics within a few generations. Studies into why this is the case have only just begun and will be subject of further work. Furthermore, the worldwide use of silver containing materials for very diverse applications should not be a major problem. In case of the observed resistance towards the copper Schiff Base, the results so far indicate that MSSA adaptation does not result in an increase in virulence factors. In the case of the zinc Schiff base P. aeruginosa was shown to be more resistant to oxidative stress caused by the ligand of ZSB.
The outcome of this project thus represents important developments that will influence human well-being, industrial processes, product development, storage and packaging for future generations. Solutions were obtained that go well beyond the state of the art and will significantly improve health care in the European Union (EU). This will ultimately lead to a substantial innovation in industry and foresees numerous perspectives for new products with higher added value in different applications. This is particularly the case in areas such as the food industry, medical and hygiene applications and textiles.
Project website: http://www.mpip-mainz.mpg.de/eu-projekte/embek1/
The central objectives of the EMBEK1 programme were to gain an improved understanding how bacteria interact and colonise surfaces, how new polymers can be made to prevent bacterial attachment and growth, and how current technologies can be used to obtain real-time, high resolution information on the nano-scale mechanisms of bacteria - surface interactions.
The project aimed to study how changes in a bacterium's genome (mutant libraries) affect the phenotypic behaviour of bacteria in terms of:
(1) their virulence;
(2) their ability to attach and colonise modified surfaces and finally,
(3) their ability to infect and kill whole organisms (insects being used as a model system for studying bacterial infection).
In order to achieve this, nano-composite and nano-structured polymeric films were to be developed using specially designed precursor molecules to create surfaces which will both directly affect a bacterium's ability to attach and will also supply antimicrobial components to kill those bacteria that do succeed in attaching to the modified surface(s). Since some of the surfaces were to be developed for clinical applications, mammalian cytotoxicity and antibacterial persistence were key issues to be studied.
Novel nano-scale material science and physical chemistry techniques were used to create and understand antimicrobial surfaces. Sophisticated molecular microbiology techniques were used to understand the genetic components governing the interaction of a bacterial biotic cell surface with the novel antimicrobial surfaces. Furthermore, the phenotypic differences of bacteria exposed to the novel antimicrobial materials were investigated by comparative proteome analysis. This duel systems approach allowed us to theoretically model the processes of bacterial attachment and survival, which in turn enabled us to improve these surfaces in an iterative approach.
Project results:
In accordance with the goals set out in Annex 1, EMBEK1 created foreground in four main areas:
(A) Materials: The development of materials and in depth analysis of properties with feedback from biological studies and modelling
Foreground: Improved knowledge in surface engineering
A catalogue of new materials including metal ion release systems with controllable release properties showing different levels of sensitivity, stability, functionality and longevity. Alternative materials that include antimicrobial nano-particles embedded in polymer thin films, bio-responsive vesicles and surface immobilised bacteriophages that respond to pathogenic bacteria only. Several publications, follow-up project (FP7-2009, Grant Agreement No. 245500)
(B) Methods: The optimisation of current analytical technologies and development of new technologies to obtain real-time, high resolution information on the nano-scale mechanisms of bacteria
Foreground: Improved knowledge on design of experiments and techniques
Establishing standard operating procedures for antimicrobial- and cytotoxicity testing. New impedimetrically based sensor to sense pathogenic bacteria. New advanced protocol vor simultaneous microevolution of resistances and determination of MIC. Nano-LC/MALDI for total proteome analysis. Theoretical and biological modelling on different levels that feed back into materials development. Several publications in print, others in preparation.
(C) Biology: To improve our understanding of how bacteria colonise surfaces using sophisticated theoretical and biological modelling and optimise current analytical technologies to obtain real-time, high resolution information on the nano-scale mechanisms of bacteria
Foreground: Improved knowledge with results indicating:
- that the pathogenic activity of P. asymbiotica in an insect relates to that in a mammal;
- that the evolution of silver ion resistance in P. aeruginosa and S. aureus is highly unlikely - as shown by the inability to develop mutant libraries over the course of the project;
- that evolution of copper Schiff's Base resistance in S.aureus and P. aeruginosa is possible and first insights into mechanisms have been achieved;
- that proteins and small molecules (such as Glutathione) in normal bodily fluids are able to protect the bacteria by binding to free silver, copper and zinc in solution, thus possibly reducing the antimicrobial effect of the metal;
- the impact of mutations in attachment and biofilm formation in insect model infections.
(D) Feasibility: Demonstrators and prototypes exploring the feasibility of the solutions
Foreground: Methods for coating different 3-dimensional (3D) implants and textile materials. New collaborations, first in-vivo tests planned for selected materials, ethics applications in progress, projects in planning, patents, potential for licensing.
As such the project EMBEK1 has fulfilled all of the goals proposed in Annex 1.
Work during the project was carried out by a total of 46 researchers over the time of 3 years. We have published 12 papers in peer reviewed journals and anticipate at least 6 others to be submitted in the near future. Three patents were filed from efforts out of the EMBEK1 project and a follow up project (BACTERIOSAFE, Grant Agreement No. 245500) was started in July 2010 with science and technology (S&T) development specifically for wound management. Smaller collaborative projects have been initiated with experts at other institutions and companies.
Potential impact:
EMBEK1 succeeded in developing a catalogue of new materials based on nano-composite, metal release systems that could be tailored in their antimicrobial efficacy, longevity of properties, stability and cytocompatibility. EMBEK1 explored the currently developing area of using nanosized and microsized biocompatible containers in the form of vesicles, which can be loaded with a desired drug or antimicrobial agent and release this upon a biological stimulus. The programme has created a large number of materials that show promising results for applications mostly as short term or single-use items. Examples of where this would be of relevance includes a number of implant devices where infection is a threat during the first 10 - 14 days of implantation, a variety of hygiene articles, diagnostic kits and one-way articles of medical clothing. In this context selected coatings were already transferred to a pre-pilot scale plant and first textile materials have been tested.
EMBEK1 developed not only antimicrobial materials and coatings, but also coatings with multiple functionalities, where antimicrobial activity is combined with cell adhesion and proliferation; or antimicrobial activity with anti-adhesive properties without cytotoxicity. Implants and devices coated with such materials offer the unique and currently not commercially available possibility to ensure reduced infection with optimum bio-integration of the implant. This work has led to a patent application by the EMBEK1 partner and will be subject of further studies and exploitation in the future.
Biological studies with the nano-composite metal release systems developed in EMBEK1 revealed highly relevant data that may influence the use and further development of silver, zinc, copper as well as phage containing wound bandages, devices, creams and the like. The work has shown, for example, that at relatively low release dose into a wound (or the environment) silver is more effective towards P. aeruginosa, while zinc is more effective towards S. aureus. At higher dosage silver is effective towards all bacterial strains tested during the work, which supports results from the literature and is in line with the currently frequent use of silver as an antimicrobial agent in wound healing. The higher dosage of silver is however cause for concern, because of issues of toxicity and reduced cell differentiation in wound healing as recently reported in the literature.
EMBEK1 investigated the response of the whole organism (insect model) towards silver and selected silver containing materials developed in EMBEK1. Sophisticated methods of proteomics and gene sequencing were used to study the effect of silver, zinc and copper release systems towards different bacteria (Photorhabdus asymbiotica, Staphylococcus aureus and Pseudomonas aeruginosa) with the aim to understand mechanisms of resistance. In this context, the complete genome of Photorhabdus asymbiotica was decoded and has been made available to academia worldwide for future studies via the UNEXE (as well as the EMBEK1) website. Tests suggest that the sequencing results for Photorhabdus asymbiotica is equally relevant for studies on S. aureus and P. aeruginosa and will be of tremendous importance in upcoming follow up projects.
In several independent studies by the different groups in EMBEK1, it was shown that S. aureus and P. aeruginosa seems to be unable to evolve resistance towards silver. Over a period of 18 months EMBEK1 scientist could not evolve mutant libraries using silver, or silver containing materials. This result is in good agreement with the literature where resistances against silver coatings is barely described and is of tremendous importance to society, to product development and industrial processing, in particular in light of the rapid spread of MRSA and the ability of bacteria, such as P. aeruginosa and S. aureus, to develop resistance towards antibiotics within a few generations. Studies into why this is the case have only just begun and will be subject of further work. Furthermore, the worldwide use of silver containing materials for very diverse applications should not be a major problem. In case of the observed resistance towards the copper Schiff Base, the results so far indicate that MSSA adaptation does not result in an increase in virulence factors. In the case of the zinc Schiff base P. aeruginosa was shown to be more resistant to oxidative stress caused by the ligand of ZSB.
The outcome of this project thus represents important developments that will influence human well-being, industrial processes, product development, storage and packaging for future generations. Solutions were obtained that go well beyond the state of the art and will significantly improve health care in the European Union (EU). This will ultimately lead to a substantial innovation in industry and foresees numerous perspectives for new products with higher added value in different applications. This is particularly the case in areas such as the food industry, medical and hygiene applications and textiles.
Project website: http://www.mpip-mainz.mpg.de/eu-projekte/embek1/