Title Promoter Affiliations Abstract "Towards mechanistic understanding of biofilms: understanding the transport phenomena, kinetics and rheology of biofilm formation and morphology." "Christian Clasen" "Soft Matter, Rheology and Technology (SMaRT)" "Biofilms are bacterial communities of cells enclosed in a self-produced matrix of extracellular polymeric substances. From an engineering perspective, it is intriguing how the bacterial colonies shape their surroundings to provide protection from external influences (mechanical/chemical stress). On the other hand, the biofilm needs to be structured in such way that bacteria can move during its formation and nutrients can reach the bacteria. In many applications, biofilms are undesired causing issues in wound healing, food production and industrial fouling. However, biofilms are of interest as a positive instrument in a growing number of biotechnical applications including bioremediation, biofertilization and energy production. The goal of this project is to extend the state of the art of the fundamental understanding of biofilm formation, morphology, functioning and dynamic behavior. The kinetics of biofilm formation, the transport phenomena inside biofilms, the mechanical properties of biofilms and bacterial motility in biofilms will all be investigated. This mechanistic view of biofilm formation is novel, and we believe the newly generated insights will contribute to methodologies and principles for design and engineering of biofilms. This will allow optimization of existing applications and will lay the groundwork for invention of new positive applications of biofilms, contributing in the end to the sustainability of our society." "Insight in methicillin-resistant Staphylococcus aureus (MRSA) biofilms: identification of key determinants in biofilm formation of highly pathogenic and globally successful MRSA clones" "Surbhi Malhotra" "Laboratory of Medical Microbiology (LMM), Vaccine & Infectious Disease Institute (VAXINFECTIO)" "Since two decades, methicillin-resistant Staphylococcus aureus (MRSA) has become a major cause of medical device-associated and postsurgical wound infections in hospitals and of pneumonia in the community. In these infections, MRSA favors the biofilm phenotype, living in a community encased in an extracellular matrix that affords protection against the host immune system and antibiotics, making these infections recalcitrant to treatment. Our laboratory has shown that two highly pathogenic and globally successful MRSA clones, USA300 and EMRSA-15, are prolific biofilm formers. Interestingly, our transcriptomics data has revealed spectacularly different mechanisms of biofilm formation between these two clones. For instance, in USA300-S391 biofilms, Hfq, a global regulator of small non-coding RNAs which in turn control rapid bacterial virulence gene expression as well as mecR, which regulates the expression of ß-lactam resistance conferring mecA gene, were both found to be highly upexpressed. EMRSA-15 biofilms, however, were not found to be MecR- or Hfq-dependent, but instead showed upexpression of multiple prophages. This fundamental project aims to dissect the role of mecR, Hfq, and prophages in mediating biofilm formation in USA300 and EMRSA-15. Identifying genes regulated by these key determinants could be better alternatives for biofilm disruption or additive therapies to antibiotics that are currently ineffective against MRSA." "Insight in methicillin-resistant Staphylococcus aureus (MRSA) biofilms: identification of key determinants in biofilm formation of two highly pathogenic and globally successful MRSA clones, USA300 and EMRSA-15." "Surbhi Malhotra" "Vaccine & Infectious Disease Institute (VAXINFECTIO)" "Since two decades, methicillin-resistant Staphylococcus aureus (MRSA) has become a major cause of medical device-associated and postsurgical wound infections in hospitals and of pneumonia in the community. In these infections, MRSA favors the biofilm phenotype, living in a community encased in an extracellular matrix that affords protection against the host immune system and antibiotics, making these infections recalcitrant to treatment. Our laboratory has shown that two highly pathogenic and globally successful MRSA clones, USA300 and EMRSA-15, are prolific biofilm formers. Interestingly, our transcriptomics data has revealed spectacularly different mechanisms of biofilm formation between these two clones. For instance, in USA300-S391 biofilms, Hfq, a global regulator of small non-coding RNAs which in turn control rapid bacterial virulence gene expression as well as mecR, which regulates the expression of ß-lactam resistance conferring mecA gene, were both found to be highly upexpressed. EMRSA-15 biofilms, however, were not found to be MecR- or Hfq-dependent, but instead showed upexpression of multiple prophages. This fundamental project aims to dissect the role of mecR, Hfq, and prophages in mediating biofilm formation in USA300 and EMRSA-15. Identifying genes regulated by these key determinants could be better alternatives for biofilm disruption or additive therapies to antibiotics that are currently ineffective against MRSA." "‘Bisceps’ Biofilm susceptibility sensors for in situ biofilm monitoring." "Hans Steenackers" "Locomotor and Neurological Disorders, Food and Microbial Technology (CLMT), Microbial and Plant Genetics (CMPG), IMEC-Interuniversitair Micro-Electronica, Universiteit Gent" "Biofilms are-surface associated microbial communities that are highly tolerant against antimicrobials and therefore cause persistent contaminations and infections in industrial and medical sectors. In particular, biofilms formed on medical implants often lead to chronic infection and implant replacement, whereas biofilms in food and other industries can lead to product contamination. This project aims at the development of a novel sensor technology, based on impedance measurement by microelectrode arrays, that allows for in vivo and in situ biofilm detection and monitoring. Recent evidence collected at KU Leuven and Imec suggests that this technology has potential not only to detect biofilms and notify in time that antimicrobial treatment is required, but also to measure biofilm structure, inform on the most effective type of antimicrobial treatment and monitor the clearance process. Since this technology is based on microelectrodes, cost-effective implementation in a broad variety of applications should be feasible. As such, successful development of this technology is anticipated to largely contribute to solving the biofilm problem. The first project pillar focuses on studying mechanisms by which biofilm formation affects impedance and the application of supervised machine learning to predict effective antimicrobial treatment protocols based on impedance data. The second pillar focuses on sensor optimization and implementation in demonstrator devices. The final pillar aims at delivering strong proof-of-concept, by applying the technology to in vivo biofilm monitoring on medical implants (rabbit model) and in situ application in food production installations (pilot-scale installations and industrial validation cases). Finally, also the generic potential of the sensor will be evaluated." "Visualisation and moleculair monitoring of the biofilm formation process, and of the impact of different biofilm inhibitation methods" "Mario Vaneechoutte" "Department of Clinical chemistry, microbiology and immunology" "Biofilm formation and the impact of different inhibition methods, which could be directed towards the quorum sensing process (asRNA silencing, enzymatic breakdown or antagonists) or directed towards the biofilm-structure (lytic phages) will be monitored by different methods." "Investigation of the PhoPQ system and nucleotide biosynthesis as targets for inhibition of Salmonella Typhimurium biofilm formation." "Jos Vanderleyden" "Centre of Microbial and Plant Genetics" "Important human pathogens become (multi-)resistant to currently availabe antibiotics, urging the need for alternative anti-bacterical treatments. Biofilms are surface-associated communities of bacteria, embedded in a self-produced slime layer. The importance of biofilms becomes clear from the fact that they are involved in 80% of all infections and occur widespread in the environment. Bacteria in biofilms are up to 1000-fold more resistant to antibiotics, disinfectants and host immune systems and are an important obstacle in the eradication of bacterial infections and bacterial contamination in industrial settings. As such, interference with biofilm formation is a promising alternative anti-bacterial strategy. Salmonella is a major foodborne pathogen, causing about 1.3 billion infections every year. The spread of Salmonella is largely due to the fact that Salmonella is able to form biobilms on different surfaces such as plants, intestinal tissue, gallstones and industrial and sanitary installations. The goal of this project is the further exploration and optimization of inhibitors of Salmonella biofilms, identified during my Ph.D. This includes a detailed study of (i) the molecular targets of the biofilm inhibitors (PhoPQ-system and nucleotide biosynthesis), (ii) the interaction of the biofilm inhibitors with these targets and (iii) the identification of new inhibitors affecting the same targets." "Identification of novel surface-expressed factors mediating virulence and biofilm formation in methicillin-resistant Staphylococcus aureus" "Herman Goossens, Jean-Pierre Hernalsteens" "University of Antwerp, Department of Bio-engineering Sciences, Biology" "Methicilline-resistant Staphylococcus aureus (MRSA) recently became of the leading causes of hospital-acquired infections (HA) and infections that are acquired in the Community (CA). HA-MRSA and CA-MRSA show major differences in the distribution of toxin and antibiotic resistance genes. The worldwide spread of some HA-MRSA clones may be associated with an increased expression of virulence genes in these bacteria. In addition to a more aggressive virulence, also a different survival strategy is used by these bacteria that in vivo produce biofilms contributing to the long-term persistence of infections. The biofilm environment provides not only more resistance to the few antibiotics that are still active against MRSA in planktonic form, but also induces a modified growth, metabolic activity and gene expression in comparison with their planktonic counterparts. The latest molecular techniques and animal models of infection will be used to investigate the differences in the formation of biofilms between HA and CA-MRSA and to study their content and expression of virulence genes. In addition, the (virulence) mechanisms which are at the basis of the success of the dominant HA-MRSA clones will be studied. The presence of new virulence factors which are brought to the expression to the surface of MRSA and could be involved in bio-film formation and in host recognition will also be examined. These studies will contribute to a better understanding of the pathogenic mechanisms used by MRSA causing recalcitrant infections and in the development of more targeted therapies for MRSA infections." "Analysis of tolerance mechanisms of biofilm yeast cells for antifugal compounds and the in vivo relevance of these mechanisms." "Bruno Cammue" "Centre of Microbial and Plant Genetics" "Among the pathogenic fungi, Candida albicans is most frequently associated with biofilm formation and is a major cause of device-related infections in most nosocomial diseases. Those infections are particularly serious because biofilm-associated Candida cells are resistant to a wide spectrum of antifungal drugs and the current treatment options for fungal biofilm-related infections are very scarce. The basis of thisdrug resistance is not clear but could be due to a combined actionof different mechanisms including (i) expression of resistance genes, (ii) drug binding to the extracellular matrix, (iii) the change in membrane composition or (iv) the presence of persister cells (cells that can survive high doses of an antimicrobial agent). Therefore, it is necessaryto search for new approaches for the treatment of these biofilm-relatedinfections.In this research project, the major aim was to gain moreinsight into tolerance mechanisms of the human pathogen C. albicans to antifungal compounds, with a focus on biofilm cells, andtranslation of this information to the in vivo situation. Knowledge of tolerance mechanisms of yeast cells to antifungal compounds is of great importance for antifungal therapy: the activity of an antifungal compound can potentially be increased by the use of the compound in combination with an inhibitor of its tolerance mechanism. The first part of this work was focused on unraveling the mode of actionand tolerance mechanisms of various antifungals against planktonic yeast cultures. More specifically, the mode of action and tolerance mechanisms of planktonic Saccharomyces cerevisiae cells to miconazole and a piperazine-1 carboxamidine derivative BAR0329 was unraveled.Miconazole and BAR0329 induce the formation of reactive oxygen species (ROS) in susceptible yeast species and are active against C. albicans planktonic and biofilm cells. Intracellular accumulation of both antifungal compounds in S. cerevisiae is dependent on functional lipid rafts. Moreover, we could demonstrate that BAR0329 induces caspase- and mitochondrial fission-dependent apoptosis in yeast. The second part of this work was dedicated toward gaining more insight in the mode of action and tolerance mechanisms of yeast biofilm cells to conventional ROS-inducing antimycotics like miconazole, amphotericin B and caspofungin. This research lead to the findings that superoxide dismutases (SODs) play an important role in the persistence and tolerance of C. albicans biofilms to miconazole and amphotericin B. This opens up the possibility for novel anti-biofilm therapy, consisting of the combination of a ROS-inducing antifungal with specific SOD inhibitors. Further investigation revealed that the transcription factor Efg1, which is a centralregulator of numerous cellular processes in C. albicans, is involved in tolerance mechanisms of C. albicans biofilms to miconazole,amphotericin B and caspofungin. Phenocopying the EFG1 deletion bydiclofenac treatment confirmed the role of Efg1 in tolerance mechanismsof C. albicans biofilms to caspofungin. In summary, the results of this doctoral research contributed to a better fundamental understanding of the tolerance mechanisms of planktonic and biofilm yeast cells against different classes of antifungals. The rational design of specific inhibitors of cellular determinants (like Efg1 and SODs) underlying biofilm tolerance to conventional antimycotics could lead to novel anti-biofilm therapy combining an antifungal compound with such inhibitor. In this respect, the in vivo data of this research project provides clear evidence that modulation of the activity of biofilm tolerance determinants, e.g. via diclofenac, is useful in combination therapy withantifungals like caspofungin to treat C. albicans biofilm-associated infections.  " "Next generation diagnostics and susceptibility testing in biofilm-related prosthetic joint infections based on a better understanding of biofilm biology – an innovative translational approach" "Tom Coenye" "Department of Diagnostic Sciences, Department of Human Structure and Repair, Department of Pharmaceutical analysis, KU Leuven" "There is growing evidence that bacteria form biofilm aggregates in synovial fluid (SF) and surface-attached biofilms on prostheses. If we want to study these biofilms in the context of prosthetic joint infections (PJI) we need better in vitro models. We also need better tools to detect and isolate relevant pathogens from PJI samples as well as novel approaches to better predict clinical success of an antimicrobial treatment. Our preliminary data show that it is possible to develop a synthetic synovial fluid (SSF) model that will support formation of biofilm aggregates by PJI pathogens and we have also shown that biofilm-based susceptibility testing is possible. In the present proposal we aim to build upon these promising preliminary data to obtain a better understanding of PJI biofilms and to translate these findings to innovative and clinically applicable approaches for diagnosis of PJI and susceptibility testing of the pathogens involved. To this end we propose the following specific objectives: -Development and validation of an in vitro SSF biofilm model that will increase our fundamental understanding of PJI biofilms and can be applied in clinical practice; -Development of SSF-based isolation and susceptibility testing protocols and comparing their performance to the current state-of-the-art in clinical practice; and -Identification of the most cost-efficient workflow for sampling and diagnosing PJI in the context of a clinical microbiology lab." "In situ Biofilm" "Frederik Leys" "Department of Electronics and information systems" "Biofilms are-surface associated microbial communities that are highly tolerant against antimicrobials and therefore cause persistent contaminations and infections in industrial and medical sectors. In particular, biofilms formed on medical implants often lead to chronic infection and implant replacement, whereas biofilms in food and other industries can lead to product contamination. This project aims at the development of a novel sensor technology, based on impedance measurement by microelectrode arrays, that allows for in vivo and in situ biofilm detection and monitoring. Recent evidence collected at KU Leuven and Imec suggests that this technology has potential not only to detect biofilms and notify in time that antimicrobial treatment is required, but also to measure biofilm structure, inform on the mosteffective type of antimicrobial treatment and monitor the clearance process. Since this technology is based on microelectrodes, costeffective implementation in a broad variety of applications should be feasible. As such, successful development of this technology is anticipated to largely contribute to solving the biofilm problem. The firstproject pillar focuses on studying mechanisms by which biofilm formation affects impedance and the application of supervised machine learning to predict effective antimicrobial treatment protocols based on impedance data. The second pillar focuses on sensor optimization and implementation in demonstrator devices. The final pillar aims atdelivering strong proof-of-concept, by applying the technology to in vivo biofilm monitoring on medical implants (rabbit model) and in situ application in food production installations (pilot-scale installations and industrial validation cases). Finally, also the generic potential of the sensor will be evaluated. "