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Projects / Programmes source: ARIS

NEW ANTIMICROBIAL STRATEGIES IN PREVENTION OF BIOFILM FORMATION BY USING LECTINS THAT INHIBIT BACTERIAL ADHESION

Research activity

Code Science Field Subfield
4.03.07  Biotechnical sciences  Plant production  Technology of vegetal origin food 

Code Science Field
T430  Technological sciences  Food and drink technology 

Code Science Field
4.01  Agricultural and Veterinary Sciences  Agriculture, Forestry and Fisheries 
Keywords
adhesion, Listeria, biofilm, lectin, mushroom, mechanism, antimicrobial activity, microfluidics, adhesin, foodborne pathogen, food infection
Evaluation (rules)
source: COBISS
Researchers (17)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  24257  PhD Aleš Berlec  Pharmacy  Researcher  2019 - 2022  225 
2.  25126  PhD Črtomir Donik  Materials science and technology  Researcher  2019 - 2022  317 
3.  33406  PhD Nikolaja Janež  Biochemistry and molecular biology  Researcher  2020 - 2022  86 
4.  11548  PhD Barbka Jeršek  Animal production  Researcher  2019 - 2022  423 
5.  54963  Blaž Jug  Biotechnology  Researcher  2020 - 2022  32 
6.  22491  PhD Anja Klančnik  Animal production  Researcher  2019 - 2022  395 
7.  18475  PhD Aleksandra Kocijan  Materials science and technology  Researcher  2019 - 2022  256 
8.  22454  PhD Tadej Kokalj  Interdisciplinary research  Researcher  2019 - 2022  76 
9.  36596  PhD Milica Perišić Nanut  Biotechnical sciences  Researcher  2019 - 2022  147 
10.  51651  PhD Tina Vida Plavec  Biotechnology  Researcher  2019 - 2022  52 
11.  37481  PhD Mateja Prunk  Biotechnology  Researcher  2019 - 2021  53 
12.  23576  PhD Jerica Sabotič  Biochemistry and molecular biology  Head  2019 - 2022  323 
13.  51713  PhD Emanuela Senjor  Biotechnology  Junior researcher  2019 - 2022  55 
14.  39997  PhD Meta Sterniša  Veterinarian medicine  Researcher  2019 - 2022  133 
15.  15600  MSc Maja Šimaga    Technical associate  2019 - 2022 
16.  55683  Tadeja Tumpej  Biotechnical sciences  Researcher  2021 - 2022  10 
17.  56053  Tanja Zupan    Technical associate  2021 - 2022 
Organisations (3)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0106  Jožef Stefan Institute  Ljubljana  5051606000  91,855 
2.  0206  Institute of Metals and Technology  Ljubljana  5051622000  6,101 
3.  0481  University of Ljubljana, Biotechnical Faculty  Ljubljana  1626914  67,219 
Abstract
The proposed project will develop an alternative approach to limit the increasing microbial resistance to antibiotics by fighting bacterial infections via prevention of the first contact of bacteria to surfaces, and thus preventing bacterial adhesion and further biofilm formation. This project will focus on Gram positive foodborne pathogenic bacteria Listeria monocytogenes as a model organism to analyse mechanisms of adhesion through specific glycan-binding proteins, i.e. the lectins (both endogenous and exogenous) as bacterial surface glycans greatly influence the interaction of bacteria with any surface. Another objective of the proposed project is to develop an improved method to study bacterial adhesion and biofilm formation using fluorescence or luminescence as part of a microfluidic platform that will enable early biofilm detection. L. monocytogenes causes listeriosis, which is the most severe zoonotic disease in the EU because of very high fatality rate. It is transmitted through consumption of contaminated food. As it is able to survive and grow in acidic, salty and cold conditions, it can colonize food processing environments very successfully and is thus regularly found on ready-to-eat foods, dairy foods, raw vegetables and fruits. The incredible persistence of L. monocytogenes is evident from the latest outbreaks in the EU, which were two multi-country outbreaks ongoing since 2015. The first step in biofilm formation is adhesion of planktonic bacteria to a surface followed by cellular proliferation and production of extracellular polymeric substances (EPS). Unlike in biofilms of many other bacteria where polysaccharides predominate, in the extracellular matrix of L. monocytogenes biofilms proteins predominate and are thus important for biofilm development and maintenance. Therefore, the proposed project will focus mainly on proteins within the extracellular polymeric substances. Our preliminary data have shown that several isolated fungal lectins can affect adhesion of bacteria to polystyrene. The project workflow will include preparation of functional recombinant lectins from higher fungi (WP1) and concurrent development of improved methods (WP2) to study bacterial adhesion and biofilm formation using engineered bacteria and a microfluidic platform enabling time-lapse and confocal microscopy. Using these newly developed improved methods we will evaluate the effects of fungal lectins on adhesion (WP3) to abiotic and biotic surfaces in static and dynamic biofilms. This will be followed by identification of target and other molecules involved in adhesion and biofilm formation (WP4) including analysis of extracellular polymeric substances and biofilm architecture. Molecules targeted by exogenous lectins will be isolated using lectin affinity chromatography and endogenous lectins will be identified by glycan competition and pull-down. Finally, the adhesion process mechanisms will be defined (WP5) by target molecule validation and new strategies to prevent surface contamination by Listeria will be proposed. The proposed project will be realized by cooperation of three research groups that have access to all of the necessary equipment to successfully finish the proposed project within 3 years. The group from Jožef Stefan Institute (IJS) led by Dr. Jerica Sabotič will coordinate the project (WP6) and provide expertise in protein characterization, microscopy and mammalian cell biology. The group from the same Department led by Assist. Prof. Aleš Berlec has expertise in engineering of G+ bacteria. The microfluidic-based method will be developed by the research group led by Dr. Tadej Kokalj from the Institute of Metals and Technology (IMT). The group led by Assist. Prof. Anja Klančnik from Biotechnical Faculty at University of Ljubljana (BF) will provide expertise in biofilm development, antimicrobial resistance mechanism, and bacterial communication.
Significance for science
The proposed project directly addresses the EC Joint Programming Initiative on Antimicrobial Resistance (AMR) as our main objective will benefit development of alternative microbial control strategies. These are critically needed for Listeria monocytogenes since listeriosis was reported by EFSA as the most severe zoonosis in the EU in 2017 with the highest hospitalization and mortality rate. To benefit the fields of biotechnology and microbiology an improved method to study bacterial adhesion will be developed using bioengineered bacteria enabling direct detection during early steps of biofilm development. Moreover, introducing the use of microfluidic techniques to study dynamic biofilms will broaden their applicability. These improved methods will have great potential to be used for studies of other foodborne pathogenic bacteria both G+ (e.g. Bacillus, Staphylococcus) and G– (Salmonella, Campylobacter, E. coli, Yersinia). This project will provide invaluable new insights into the initial steps of bacterial biofilm formation by identification of new glycan-binding proteins involved in the process of adhesion to surfaces and/or interaction between bacteria. Furthermore, the use of lectins from higher fungi represents an original aspect of this project proposal. Only detailed understanding of the adhesion process can lead to development of new agents for use in food and medical biotechnology to prevent biofilm formation. It will enable us to propose new strategies for control and prevention of biofilm formation in the food industry and in human and veterinary medicine. We expect to get insight into the adhesion of Listeria to relevant cell types, which can provide new research directions for prevention of primary and/or secondary infection. The alternatives to antibiotics that affect adhesion have the advantage of limiting the microbial colonization in the early stages, and they therefore contribute to reduced use for antibiotics as well as AMR development since they provide little evolutionary pressure on the pathogen. Furthermore, the use of any new antimicrobial strategies identified can be extended to the control of other pathogenic Gram positive bacteria such as those from the genera Staphylococcus, Streptococcus, Bacillus. Finally, we will be working exclusively with water soluble molecules, which gives them an advantage over many antimicrobial agents with limited value due to poor water solubility. In the food production and processing sectors the knowledge gained here will be useful for the development of appropriate quantitative microbial risk assessment for the food-production chain and distribution contributing to safer food processing environment. The project will enable training of young scientists since PhD students as well as Master students will be included. Furthermore, we are involved in the initiative Metrology of Carbohydrates for glycoscience promotion in Europe, which will make our data instantly relevant to a wide audience.
Significance for the country
The proposed project directly addresses the EC Joint Programming Initiative on Antimicrobial Resistance (AMR) as our main objective will benefit development of alternative microbial control strategies. These are critically needed for Listeria monocytogenes since listeriosis was reported by EFSA as the most severe zoonosis in the EU in 2017 with the highest hospitalization and mortality rate. To benefit the fields of biotechnology and microbiology an improved method to study bacterial adhesion will be developed using bioengineered bacteria enabling direct detection during early steps of biofilm development. Moreover, introducing the use of microfluidic techniques to study dynamic biofilms will broaden their applicability. These improved methods will have great potential to be used for studies of other foodborne pathogenic bacteria both G+ (e.g. Bacillus, Staphylococcus) and G– (Salmonella, Campylobacter, E. coli, Yersinia). This project will provide invaluable new insights into the initial steps of bacterial biofilm formation by identification of new glycan-binding proteins involved in the process of adhesion to surfaces and/or interaction between bacteria. Furthermore, the use of lectins from higher fungi represents an original aspect of this project proposal. Only detailed understanding of the adhesion process can lead to development of new agents for use in food and medical biotechnology to prevent biofilm formation. It will enable us to propose new strategies for control and prevention of biofilm formation in the food industry and in human and veterinary medicine. We expect to get insight into the adhesion of Listeria to relevant cell types, which can provide new research directions for prevention of primary and/or secondary infection. The alternatives to antibiotics that affect adhesion have the advantage of limiting the microbial colonization in the early stages, and they therefore contribute to reduced use for antibiotics as well as AMR development since they provide little evolutionary pressure on the pathogen. Furthermore, the use of any new antimicrobial strategies identified can be extended to the control of other pathogenic Gram positive bacteria such as those from the genera Staphylococcus, Streptococcus, Bacillus. Finally, we will be working exclusively with water soluble molecules, which gives them an advantage over many antimicrobial agents with limited value due to poor water solubility. In the food production and processing sectors the knowledge gained here will be useful for the development of appropriate quantitative microbial risk assessment for the food-production chain and distribution contributing to safer food processing environment. The project will enable training of young scientists since PhD students as well as Master students will be included. Furthermore, we are involved in the initiative Metrology of Carbohydrates for glycoscience promotion in Europe, which will make our data instantly relevant to a wide audience.
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