Projects

Home    Projects    Accredited testing laboratory of the DBM FFBT UCT Prague

1. Detection, identification and characterization of food relevant microorganisms and food microbiome

Rapid and reliable detection, quantification and identification of microorganisms present in food are crucial requests of food microbiology and prevention of foodborne diseases. This requires accurate and fast methods, allowing also the detection of microbial contamination source, are therefore being developed. In our lab, conventional culture methods (including ISO methods), and appropriate molecular biology methods (end-point PCR, qPCR, MALDI-TOF MS, Sanger sequencing) are used.

Specific topics of our research include:

• Isolation and identification of target groups of microorganisms from food and food processing
• Development of new methods for detection, quantification and typing of microorganisms in food
• Solving microbiological problems in food practice
• The impact of protective microbial cultures on the food microbiome

Support:

• Collaboration with industrial partners
• MZE QJ1210300 - Quality and safety assurance systems for dairy products by suitable methods applicable in practice
• MZE QL24010251 Nature in Cheese - The complex microbiota of cheese - new methods to assess its composition and safety for subsequent use in cheese technology

2. Biofilm formation and resistance

Bacteria in the natural environment are predominantly found not as free-moving planktonic cells but as sessile communities on a solid substrate in the form of a biofilm. The cells of the biofilm are irreversibly attached to the surface and to each other by extracellular polymeric substances (EPS) matrix. Due to the protective matrix layer and EPS, and cellular communication, biofilm cells are highly resistant to environmental stresses (such as nutrition depletion, penetration of antimicrobials, etc.), and they also show different growth and gene expression compared to planktonic cells. In terms of food microbiology, biofilm is a major problem in production and transport facilities, as it causes corrosion of materials or contamination of products. In our lab, we focus on studying of the conditions and characteristics of biofilm formation by food pathogens, together with the structure and expression of genes regulating its formation. For practical purposes, we then study the effect of disinfectants and antimicrobials on biofilm eradication.

Specific topics of our research include:

• Qualitative and quantitative analysis of single- and multi-species biofilms of food pathogens
• Effect of antimicrobial agents (such as disinfectants, natural extracts and compounds, nanoparticles, and carbon-based materials) on biofilm formation and eradication, and transcription of biofilm formation related genes

3. Antibiotic resistance in the food chain

The increasing prevalence of bacterial resistance to antibiotics is one of the major challenges of the 21^st century. The emergence of resistant bacterial strains consequently narrows the spectrum of suitable antibiotics for the treatment and/or prevention of even common bacterial infections, e.g., in surgery. Among the most frequent sites for the emergence and/or spread of new antibiotic resistance determinants (hot spots) are wastewater treatment facilities, hospitals, and also the food chain. Therefore, we focus on developing of new rapid and reliable methods for antibiotic resistance determinants analysis.

Specific topics of our research include:

• Occurrence of resistance genes in foodborne and pathogenic bacteria (e.g., Salmonella spp., Escherichia coli, Listeria monocytogenes and Staphylococcus aureus)
• Detection of antibiotic resistance genes in the food chain and other hot spots (wastewater treatment) and study of their genetic determinants (e.g., efflux pump genes, beta-lactamases, tetracycline resistance genes)
• The food chain bacteriome and the variability of its resistome
• Validation of qPCR protocols for quantification of antibiotic resistance genes
• MALDI-TOF MS phenotypic and genotypic profiling of antibiotic resistant bacteria
• Monitoring resistance and multidrug resistance of pathogens isolated from different types of samples (food, clinical cases, wastewater treatment, etc.)

Support:

• ARG Tech - Technology for the removal of antibiotic resistance genes from sewage sludge applied in agriculture TAČR SS01020112
• Czech Platform for Antibiotic Resistance (CZEPAR) (https://czepar.vscht.cz/czepar)
• EU4 Health project-EU-WISH (Wastewater Integrated Surveillance for Public Health (https://www.eu-wish.eu/)

4. Analysis of bacteriome of activated sludge by nanopore sequencing

The bacteriome of activated sludge is very heterogeneous, with some bacterial species, usually very difficult to cultivate or not yet cultivated, but technologically undesirable and problematic for the proper treatment process. Therefore, usually microscopic analysis or various molecular biological methods are used for their determination. The nanopore sequencing of 16S rRNA amplicons and metagenomic DNA is very suitable because, at a favourable cost and with a performance suitable for routine laboratory practice, it allows both the overall analysis of the bacteriome of isolated DNA and the acquisition of information on the frequency of target technologically problematic species.

Specific topics of our research include:

Introduction and use of a modern nanopore sequencing method for the detection and quantification of technologically undesirable bacterial species present in activated sludge, in order to determine and optimize steps leading to the improvement of the wastewater treatment process.

Support:

• VS SIGMA DC2 TQ03000804 NASEK - Use of nanopore sequencing of activated sludge bacteria for wastewater treatment process control

5. Expression of virulence factors of bacterial pathogens

Staphylococcus aureus is a pathogen producing a variety of toxic substances causing various diseases. From the perspective of food microbiology, its most important characteristic is its ability to produce thermostable enterotoxins that can cause alimentary intoxication or food poisoning. We focuse on the study of the expression of genes involved in biofilm formation and enterotoxin production, both at the mRNA and protein level. At the mRNA level, we use reverse transcription and quantitative real-time PCR (RT-qPCR), and at the protein level, we use immunochemical ELISA. In addition to this work, basic phenotypic and genotypic characterisation is also performed on the strains tested. In addition to the transcription of S. aureus genes, the transcription rate of virulence factors of the opportunistic pathogen Cutibacterium acnes is also investigated

Specific topics of our research include:

• Staphylococcus aureus virulence

6. Analysis of genetically modified organisms (GMOs)

GMOs are defined as those organisms whose genetic material has been altered in a way that cannot be achieved naturally. By means of targeted genetic modifications, it is possible to achieve desired characteristics of organisms, such as increased resistance to negative influences (pests, weeds, diseases, climatic conditions), increased content of macro and micronutrients or accelerated growth without reducing nutritional value. In our laboratory, we perform routine analyses of the presence and precise quantification of GMOs, through PCR and qPCR. The monitored sequences determining transgenic DNA (promoters and transcription terminators, selection markers) as well as variety-specific border sequences of specific GMOs are analysed. Both in house procedures (SOP) and ISO CSN protocols are used.

Specific topics of our research include:

• Detection and quantification of GMOs in food
• Multiplex PCR/qPCR

Support:

• Membership and cooperation with the Czech Commission for the Management of Genetically Modified Organisms and Genetic Products
• Membership and cooperation with the Scientific Committee on Genetically Modified Food and Feed

Annual financial support from the Ministry of the Environment

7. Food adulteration

Food adulteration is a major problem in today's world market. The main reason for food adulteration is the manufacturer's desire to reduce production costs by declaring a higher quality in the consumer information on the product packaging than is actually sold. It is therefore a deception of the consumer, but at the same time it can also endanger the consumer's health. Meat and meat products are among the most expensive foodstuffs and are therefore among the most frequently adulterated commodities. One of the common ways of deceiving consumers is to substitute meat of a lower quality for meat of a lower value and to misrepresent the proportion of meat on the product label. Methods based on DNA analysis are a very accurate tool for detecting the authenticity of the raw materials from which food is produced. Our goal is to develop and validate methods that are suitable for the analysis of complex and processed food matrices. Our research focuses on species identification of animals and plants by PCR, DNA sequencing and proteomic analysis by MALDI-TOF MS. Projects are focused on molecular biological analysis of important livestock species (cattle, pig, horse or poultry), microsatellite analysis and PCR identification of poppy and species identification of fish by PCR amplifying the nuclear gene encoding the major fish allergen, parvalbumin.

Specific topics of our research include:

• Identification of food important species by DNA analysis
• Identification of fish through molecular biological and proteomic approaches
• Development of a molecular marker for the differentiation of poppy varieties

Support:

• MZe (NAZV) QK23020101: Comprehensive laboratory strategy for identification of insect species for human consumption and production of processed animal protein, authentication of insect-based foods
• MZE QK1910231 - Novel approaches to detect fish meat adulteration using genomic DNA
• MZe (NAZV) QJ1530272 - Comprehensive strategies for effective detection of food adulteration in the (pro)production-consumer chain

8. Interaction of nanofibrous materials with microorganisms

Nanotechnology has become part of our everyday life. A lot of attention has been paid to their research in various sectors, including medicine and food. Currently, there is great interest in the use of nanomaterials in tissue engineering, a field concerned with the preparation of biocompatible tissue and organ replacements, or in the production of active food packaging to extend the shelf life of food without the need for preservatives. Microbiological safety and knowledge of the interactions of microorganisms with the applied nanomaterials is essential for both of these fields. The effect of non-functionalized and functionalized nanofibrous textiles on the growth and biofilm formation of selected pathogenic and beneficial microorganisms is being studied in our laboratory. The influence of the morphology of nanomaterials softened by electrospinning of different polymers on microbial interactions is investigated. Current research shows that it is through morphology that microbial growth can be effectively suppressed or promoted. To study the interactions between microorganisms and nanofibrous materials, we use conventional microbiological methods as well as molecular biological methods and microscopic methods, especially SEM and CLSM. Nanofibrous materials are prepared and characterized by collaborators from Czech companies and the Technical University of Liberec.

Specific topics of our research include:

• Effect of morphology and structure of non-functionalized nanomaterials on bacterial growth, biofilm formation and retention of selected clinical and foodborne pathogens and biotechnologically important microorganisms
• Application of nanofibers in food packaging and their effect on food microbiota
• Development of antibacterial nanofibrous surgical threads to reduce the risk of nosocomial infections

Support:

• GAČR 23-05154S Interaction of prokaryotic and eukaryotic cells with nanofibers of different morphology and structure
• PO, OP PIK APLIKACE, CZ.01.1.02/0.0/0.0/16_084/0009936 - Application of nanofibres in food packaging

9. Analysis of viral RNA/DNA in food and water

Foodborne diseases can be caused by the ingestion of food in which pathogenic microorganisms or toxins produced by them are present. Microorganisms contaminating food and water include not only many bacteria and fungi, but also viruses. The most common viruses transmitted through food and water are noroviruses and hepatitis A virus. Less common are rotaviruses, hepatitis E virus, astroviruses, enteroviruses, coronaviruses, parvoviruses and adenoviruses. In our laboratory we are involved in the detection of noroviruses and SARS-CoV-2 coronavirus. SARS-CoV-2 coronavirus is a newly discovered strain from the Coronaviridae family that causes a serious respiratory disease called COVID-19. As of late 2019, the virus has caused a global pandemic. Given the global public health emergency, it is important to be able to quickly and reliably detect this virus not only as a sign of disease in an infected person, but also from a variety of materials (e.g., food packaging) or wastewater. This would allow monitoring of the presence of the virus and better prevention of further zoonotic events of a similar nature.

The specific topics we are addressing in the project are:

• Detection of noroviruses in food and water
• Detection of SARS-CoV-2 RNA and monkeypox virus (Mpox) in water and sludge
• Detection of avian influenza RNA

Support:

• MZE (NAZV) QL24010383: Early detection of sources of avian influenza infection by detection of viral particles in environmental samples
• TA ČR SS01020112 - Technology for the removal of antibiotic resistance genes from sewage sludge applied in agriculture

10. Interaction of graphene and graphene-containing composite materials with microorganisms

In recent decades, graphene materials have attracted the attention of basic and applied research, which, in addition to their ability to improve the physical and chemical properties of other matrices, show promising antimicrobial effects. The mechanisms by which graphene interacts with microbial cells and how different forms of graphene differ in their effects are still not fully understood. As part of our research in collaboration with the Institute of Inorganic Chemistry at the University of Technology, we are investigating the effect of graphene materials on the growth and biofilm formation of biotechnologically important microorganisms and microbial communities. The research also includes genotoxicity assessment. Graphene materials of different spatial conformations are included in the testing: 1D (nanotubes), 2D (e.g., graphene, graphene oxide) and 3D (composites and nanofibers containing 1D and 2D materials). To study the interactions between microorganisms and graphene materials, we use classical microbiological methods as well as molecular biology methods and microscopy methods, in particular SEM and CLSM

The specific topics we are addressing in the project are:

• Effect of graphene materials on the growth and biofilm formation of selected microorganisms
• Study of the mechanism of action of graphene materials on microbial cells - effect on metabolic activity of cells, production of reactive oxygen species, etc.
• In vitro and in vivo genotoxicity of graphene materials

Support:

• OP JAK CZ.02.01.01/00/22_008/0004631 Materials and Technologies for Sustainable Development (MATUR)

11. Methods of biological control of potatoes

The use of antimicrobial activity of bacteria and fungi is one of the possible ways of biological protection of potatoes against the effect of pectinolytic bacteria (Pectobacterium spp. and Dickeya spp.) causing soft rot of tubers. The most common producers of antimicrobial molecules are members of the genera Pseudomonas, Bacillus, Serratia, Lactobacillus or Lactococcus. These bacteria are capable of producing a wide range of substances with antimicrobial activity, such as hydrogen peroxide, antibiotics, bacteriocins, siderophores, or various volatile organic compounds. It is known that some bacteria can also inactivate signaling molecules in the quorum-sensing process, thus affecting the expression of virulence factors of phytopathogenic bacteria. These are mainly bacteria belonging to the genera Delftia, Ochrobactrum, Bacillus and Rhodococcus. The strategy of searching for suitable bioagents can be very simply summarized in the following steps: (i) isolation of microbial population; (ii) verification of antimicrobial activity on target phytopathogens under laboratory conditions; (iii) elimination of isolates with intrinsic pectinolytic activity. This baseline screening is then followed by experiments aimed at monitoring the effect of biogens directly on potato tubers under greenhouse conditions and subsequently in semi-field conditions.

Specific topics of our research include:

• Isolation of soil microbial populations from sites in the Czech Republic.
• Verification of antimicrobial activity on target phytopathogens
• Determination of pectinolytic activity
• Identification of fungal and bacterial isolates (morphology, biochemical tests, MALDI-TOF, sequencing of suitable DNA loci)

Support:

• QL24010148 alternative methods of biological control of potato using bioagents and substances of natural origin

12. Biologically active substances

Due to the increasing number of antibiotic resistant microorganisms, it is necessary to search for new substances with antimicrobial activity. These can be obtained either by isolation from a natural source, by chemical synthesis using structural modification or by de novo synthesis. Plants produce a large number of natural products that can be applied to promote human health. Drugs based on natural products have traditionally been administered orally as crude extracts, but this raises health concerns due to harmful or undesirable intermediates in the crude extracts. Technological advances, however, have created the conditions for more sophisticated and higher quality production of pharmaceutical natural products.

Specific topics of our research include:

• Antimicrobial activity, cytotoxicity, anti-inflammatory activity of plant extracts
• Evaluation of extraction methods in the processing of plant material according to their biological activities
• Synthesis of new biologically active substances based on peptides and/or triterpene compounds

Support:

• TA ČR Project No. TN02000044 - BIOCIRKL - Biorefining and circular economy for sustainability

13. Endophytic microorganisms

Due to the increasing world population, there is pressure for sufficient and stable agricultural production. Crop cultivation is severely limited by adverse environmental conditions, pests and diseases, which are currently compounded by climate change. Pesticides are therefore used to ensure sufficient crop yields. Although the application of these chemicals is very effective, it is associated with many pitfalls concerning the sustainability of this crop production system. Pesticides are chemicals that have an adverse impact on the environment, including humans. Thus, modern trends in agriculture seek to reduce the use of pesticides and find new and environmentally friendly solutions to the problematics of crop protection. Endophytes are becoming an important element in biotechnological applications due to the production of various enzymes and other biologically active substances. They are already being used in agriculture, medicine, food and cosmetic industries, but they can also be useful in other fields.

Specific topics of our research include:

• Isolation and identification of endophytic microorganisms
• Characterization of their biochemical activities
• Application experiments on plants

Support:

• TA ČR project No. TN02000044 - BIOCIRKL - Biorefining and circular economy for sustainability

14. Circular economy - preparation of bio-concrete

The Paris Climate Agreement and the Green are currently key efforts to reduce greenhouse gas emissions, including carbon dioxide. Among the main emitters is the construction industry, with a significant proportion of CO₂ produced in the production of Portland cement, used for concrete production. One way to reduce cement production is to introduce recycling of waste building materials. Our group is working on recycling finely crushed waste concrete produced during demolition, while using microorganisms that are able to precipitate calcium carbonate (MICP - microbially induced calcium carbonate precipitation). Under specific conditions, the microorganisms are able to form different structures of CaCO₃ crystals that bind the particles of the waste concrete together to form a solid, composite sample, called bio-concrete. The resulting composite bio-concrete samples are analysed for structural and mechanical properties in cooperation with the Faculty of Civil Engineering, CTU. Thanks to this technology, it is possible not only to reduce CO₂ emissions, but also to use waste building material. However, one of the obstacles to transferring this technology to an industrial scale is the cost of the whole process, a significant part of which is the cost of commercial cultivation media. Therefore, one of our goals is to replace commercial culture media with alternative nutrient sources, such as by-products from the poultry or brewing industries.

Specific topics of our research include:

• Validation of the ability to precipitate calcium carbonate in prokaryotic (bacteria) and eukaryotic (fungi) microorganisms.
• Modification of composite sample preparation procedures to improve their mechanical properties, for example by adding natural materials
• Isolation and identification of new microorganisms with the ability to precipitate calcium carbonate from different environmental niches, for example from the Koněpruské limestone caves
• Analysis of the composition of microbial communities in environments where calcium carbonate formation takes place
• Replacing commercial growth media with cheaper alternatives prepared from animal by-products and optimising their preparation
• Comprehensive analysis of the obtained composite bio-concrete samples - SEM, XRD, EDS, mechanical properties (stiffness, strength)
• Carbon footprint assessment and overall LCA (Life-Cycle Assessment) analysis to evaluate environmental impacts

Support:

• GAČR 22-02702S: Microbiologically induced calcite precipitation in concrete recycling for the production of materials with a negative carbon footprint
• National Centre of Competence, TAČR TN02000044: "Biorefining and circular economy for sustainability"

15. Biodeterioration of cultural monuments

Cultural heritage objects carry valuable information about the development of our society from ancient times to the present. Unfortunately, they are commonly subject to biodeterioration, which are undesirable changes caused by the activities of organisms, mainly microorganisms. This process causes irreversible damage to objects of cultural heritage and thus incalculable loss of information. It is therefore important to prevent microbial contamination of cultural heritage by storing it in appropriate climatic conditions. Where collections are already contaminated, it is essential to identify and remove the microorganisms. Our working group focuses on the identification of microbial populations on different types of cultural heritage, especially photographs and books, but also in the air, as microorganisms in the air are considered to be one of the main causes of contamination of cultural heritage. For identification, we use both a culture-dependent approach using MALDI-TOF MS or Sanger sequencing, where the emphasis is on capturing as much diversity as possible, and a culture-independent approach using amplicon sequencing with Illumina MiSeq, based on both DNA and RNA isolation. An important part of our research is testing enzyme activities, which allows us to monitor the biodeterioration potential of isolates, as well as testing the resistance of microbial isolates to disinfectants. This comprehensive approach allows us to contribute to the protection of cultural heritage from destruction caused by microbial activity.

Specific topics of our research include:

• Monitoring of microbial contamination of historical books, photographs and filmstrips in Czech depositories
• Development of a gentle methodology for the collection and isolation of bacteria, yeasts and moulds from cultural monuments
• Identification and characterisation of bacterial strains contaminating photographic and cinematographic materials
• Identification and characterisation of filamentous micromycetes isolated from archival materials
• Comparison of the identification of isolates by traditional culture and sequencing methods
• Testing the resistance of microbial isolates to disinfectants

Support:

• NAKI II (Grant No. DG18P02OVV062): Biodiversity of black and white photographic and cinematographic materials in archival collections and methods for their disinfection

16. Factors affecting the soil ecosystem

Our research group started working on this topic in 2015, in the framework of cooperation with the Faculty of Agrobiology, Food and Natural Resources of the Czech University of Life Sciences. Currently, there is a strong emphasis on sustainable agriculture and at the same time on maximum plant productivity, which is mainly influenced by soil quality. In our research we are looking at how different agronomic practices affect the soil ecosystem. One of these strategies is the application of fertilizers such as sewage sludge, manure or mineral fertilizers. The application of these fertilizers has been carried out at four sites in the Czech Republic since 1996 and the individual sites differed not only in terms of soil physico-chemical parameters but also in terms of climatic conditions. However, this strategy can affect the soil not only positively but also negatively, for example by increasing the occurrence of undesirable pathogenic microorganisms or by increasing various types of antibiotic resistance genes. Another way to improve soil quality is through the addition of biochar, which is a carbonaceous material produced by pyrolysis of organic waste. The application of biochar to soil contributes to soil carbon sequestration, improving soil properties, is an important source of nutrients, helps maintain soil moisture and promotes microbial diversity. Two types of waste were used in the project: beech wood chips and residue from the mechanical separation of chicken meat, which were prepared at two different pyrolysis temperatures. The soil ecosystem can also be affected by the presence of different types of pollutants, which are often not only present in sewage sludge or manure, but can also be present in low concentrations in water used for plant watering.

Specific topics of our research include:

• Effect of the addition of different types of fertilizers and biochar on:
• Changes in microbial communities in the soil, in the rhizosphere and on endophytic plant populations
• soil microbial activity
• the presence of pathogenic microorganisms
• increase in antibiotic resistance genes in soil
• Effect of biochar and arbuscular mycorrhiza application on the degradation, accumulation and transport of three groups of contaminants (pharmaceuticals, plasticizers and synthetic aromatic compounds) in plants and soil, rhizosphere and endophyte populations

Support:

• GAČR 16-07441S, GAČR 19-02836S