Research

The evolution of antimicrobial resistance is strongly reducing our options for antimicrobial treatment, both in agriculture, industry and medicine. At the same time regulatory demands on microbial safety, waste water limitation, and biocides have never been so strict. We urgently need new antimicrobial strategies, therefore, but most importantly we need strategies where the evolution of resistance is less rapid.

Most current antimicrobials have been designed to act against free-living bacteria. Over the last decades it became clear, however, that microbes commonly live together in dense and genetically-diverse communities. If these communities are surface-associated and embedded in a self-produced slime-layer they are generally called biofilms. Microbial communities often show a strongly increased tolerance against antimicrobials. New antimicrobial strategies, therefore, should take this community mode-of-life into account.

The MICA Lab studies microbial community behaviour and develops antimicrobials targeting communities. Our main goal is to obtain a better understanding of social interaction in microbial communities, resistance development in communities and methodologies to monitor communities in situ. This should allow us to develop novel types of antimicrobials that interfere with social interactions (socio-active strategies), resistance development (anti-resistance strategies) or take benefit of improved monitoring (observation-guided strategies). To support this research, we have set up an integrated development platform for antimicrobial compounds.

Social interactions in microbial communities
Contact
Socio-active strategies

Social interactions

We aim to understand how microbes within communities interact with each other. It is increasingly recognized that social interactions between microbes can strongly enhance the tolerance and pathogenicity of communities. Social interactions therefore present an interesting target for novel types of antimicrobials (socio-active strategies), especially because in some cases resistance development is not expected. Our current research mainly focuses on social interactions within bacterial communities containing Salmonella, within contaminating biofilms in breweries and greenhouses and within biofilms on orthopedic implants and urinary catheters.

Publications
  • Parijs, I., Steenackers, H. (2018). Competitive inter-species interactions underlie the increased antimicrobial tolerance in multispecies brewery biofilms. The ISME Journal, 12(8):2061-2075.
  • Spacova, I., Lievens, E., Verhoeven, T., Steenackers, H., Vanderleyden, J., Lebeer, S., Petrova, M.I. (2018). Expression of fluorescent proteins in Lactobacillus rhamnosus to study host-microbe and microbe-microbe interactions. Microbial Biotechnology, 11(2):317-331.
  • Lories, B., Parijs, I., Foster, K.R., Steenackers H.P. (2017). Meeting report on the ASM Conference on Mechanisms of Interbacterial Cooperation and Competition. The Journal of Bacteriology. doi: 10.1128/JB.00403-17.
  • Steenackers, H., Parijs, I., Foster, K.R., Vanderleyden, J. (2016). Experimental Evolution in Biofilm Populations; FEMS Microbiology Reviews, 40(3), 373-97.

2 publications submitted.

Resistance evolution in microbial communities
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Anti-resistance strategies

Resistance evolution

We aim to understand how resistance development in microbial communities differs from resistance development in isolated, free-living microbes. Our recent experiments indeed indicate that different aspects of the microbial community mode-of-life can promote the evolution of resistance against conventional antimicrobials. This demonstrates the potential of 'anti-resistance strategies' that interfere with community behaviour to enhance the longevity of conventional antibiotics. Our current research mainly focuses on resistance development in Salmonella biofilms, in brewery biofilms and biofilms on orthopedic implants and catheters.

Publications
  • Steenackers, H., Parijs, I., Foster, K., Vanderleyden, J. (2016). Experimental Evolution in Biofilm Populations; FEMS Microbiology Reviews, 40(3), 373-97.
  • Roberfroid, S., Vanderleyden, J., Steenackers, H. (2016). Gene expression variability in clonal populations: causes and consequences. Critical Reviews in Microbiology, 42(6), 969-84.
  • Pulido-Tamayo, S., Sánchez-Rodríguez, A., Swings, T., Van den Bergh, B., Dubey, A., Steenackers, H., Michiels, J., Fostier, J., Marchal, K. (2014). Frequency-based haplotype reconstruction from deep sequencing data of bacterial populations. Nucleic Acids Research, 43(16), e105. doi: 10.1093/nar/gkv478.
In situ monitoring of microbial communities
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Observation-guided strategies

In situ monitoring

The tolerance of a particular biofilm to antimicrobial treatment is highly dependent on its specific structure. Hence, rapid detection of biofilms and accurate monitoring of the biofilm structure, thereby predicting its tolerance to antimicrobial treatment, is of utmost importance. Today, however, no technologies exist that allow for in depth in situ biofilm characterization. In collaboration with several research partners, we aim to develop a novel sensor technology, based on impedance measurement by microelectrode arrays, that allows for in situ biofilm monitoring and prediction of its susceptibility to different types of antimicrobials.

Publications
  • Goikoetxea, E., Routkevitch, D., de Weerdt, A., Green, JJ., Steenackers, H., Braeken, D. Impedimetric fingerprinting and structural analysis of isogenic E. coli biofilms using multielectrode arrays (2018). Sensors and Actuators B: Chemical, 15 June 2018, 263:319-326. doi: 10.1016/j.snb.2018.01.188.
  • Maes, S., Heyndrickx, M., Vackier, T., Steenackers, H., Verplaetse, A., Reu, K. (2018). Identification and Spoilage Potential of the Remaining Dominant Microbiota on Food Contact Surfaces after Cleaning and Disinfection in Different Food Industries. J Food Prot. 2019 Feb;82(2):262-275. doi: 10.4315/0362-028X.JFP-18-226.

Development platform for antimicrobial compounds

To support the above described research lines, the MICA Lab is coordinating an interdisciplinary platform for the development of antimicrobials, which is based on several collaborations with research groups within and outside the KU Leuven. This platform includes in silico and in vitro screening of antimicrobial agents, chemical synthesis and structure-activity-relationship studies, mode of action studies and evaluation of resistance development, and design of combination therapies and delivery strategies. More detailed information on this platform can be found here.

Publications
Chemical synthesis and in vitro screenings of anti-biofilm compounds
  • Trang, TTT., Dieltjens, L., Hooyberghs, G., Waldrant, K., Ermolat'ev, D., Van der Eycken, E., Steenackers, H. Enhancing the anti-biofilm activity of 5-aryl-2-aminoimidazoles through nature inspired dimerisation (2018). Bioorganic & Medicinal Chemistry, 26(8):1470-1480.
  • Gill, RK., Kumar, V., Robijns, SC., Steenackers, HP., Van der Eycken, EV., Bariwal, J. Polysubstituted 2-Aminoimidazoles as Anti-Biofilm and Antiproliferative Agents: Discovery of Potent Lead (2017). European Journal of Medicinal Chemistry, 138, 152-169.
  • Yssel, A., Vanderleyden, J., Steenackers, H. Repurposing of nucleoside- and nuclebase-derivative drugs as antibiotics and biofilm inhibitors (2017). Journal of Antimicrobial Chemotherapy, 72(8):2156-2170.
  • Peeters, E., Hooyberghs, G., Robijns, S., Waldrant, K., De Weerdt, A., Delattin, N., Liebens, V., Kucharíková, S., Tournu, H., Verstraeten, N., Dovgan, B., Girandon, L., Fröhlich, M., De Brucker, K., Van Dijck, P., Michiels, J., Cammue, B.P., Thevissen, K., Vanderleyden, J., Van der Eycken, E., Steenackers, H.P. Modulation of the substitution pattern of 5-aryl-2-aminoimidazoles allows fine-tuning of their anti-biofilm activity spectrum and toxicity. Antimicrobial Agents and Chemotherapy, 60(11), 6483-6497.
  • Mishra, N.M., Briers, Y., Lamberigts, C., Steenackers, H., Robijns, S., Landuyt, B., Vanderleyden, J., Schoofs, L., Lavigne, R., Luyten, W., Van der Eycken, E.V. (2015). Evaluation of the antibacterial and antibiofilm activities of novel CRAMP-vancomycin conjugates with diverse linkers. Organic and Biomolecular Chemistry, 13(27), 7477-86.
  • Liebens, V., Gerits, E., Knapen, W., Swings, T., Beullens, S., Steenackers, H., Robijns, S., Lippell, A., O'Neill, A., Veber, M., Fröhlich, M., Krona, A., Lövenklev, M., Corbau, R., Marchand, A., Chaltin, P., De Brucker, K., Thevissen, K., Cammue, B., Fauvart, M., Verstraeten, N., Michiels, J. (2014). Identification and characterization of an anti-pseudomonal dichlorocarbazol derivative displaying anti-biofilm activity. Bioorganic & Medicinal Chemistry Letters, 24(23), 5404-8.
  • Steenackers, H., Dubey, A., Robijns, S., Ermolatev, D., Delattin, N., Dovgan, B., Girandon, L., Fröhlich, M., De Brucker, K., Thevissen, K., Balzarini, J., Van der Eycken, E., Vanderleyden, J. (2014b). Evaluation of the toxicity of 5-aryl-2-aminoimidazole-based biofilm inhibitors against eukaryotic cell lines, bone cells and the nematode Caenorhabditis elegans. Molecules, 19, 16707-16723.
  • De Brucker, K., Delattin, N., Robijns, S., Steenackers, H., Verstraeten, N., Landuyt, B., Luyten, W., Schoofs, L., Dovgan, B., Fröhlich, M., Michiels, J., Vanderleyden, J., Cammue, B., Thevissen, K. (2014). Derivatives of the mouse cathelicidin-related antimicrobial peptide (CRAMP) inhibit fungal and bacterial biofilm formation. Antimicrobial Agents and Chemotherapy, 58 (9), 5395-5404.
  • Steenackers, H., Ermolat'ev, D., Thi Thu Trang, T., Savalia, B., De Weerdt, A., Shah, A., Vanderleyden, J., Van der Eycken, E. (2014a). Microwave-Assisted One-Pot Synthesis and Anti-Biofilm Activity of 2-Amino-1H-imidazole/Triazole Conjugates. Organic and Biomolecular Chemistry, 12(22), 3671-8.
  • Robijns, S., De Pauw, B., Loosen, B., Marchand, A., Chaltin, P., De Keersmaecker, S., Vanderleyden, J., Steenackers H. (2012). Identification And Characterization Of 4-[4-(3-Phenyl-2-propen-1-yl)-1-Piperazinyl]-5H-Pyrimido[5,4-B]Indole Derivatives As Salmonella Biofilm Inhibitors. FEMS Immunology and Medical Microbiology, 65(2), 390-394.
  • Steenackers, H., Ermolatev, D., Savaliya, B., De Weerdt, A., De Coster, D., Shah, A., Van der Eycken, E., De Vos, D., Vanderleyden, J., De Keersmaecker, S. (2011). Structure-activity relationship of 2-hydroxy-2-aryl-2,3-dihydro-imidazo[1,2-a]pyrimidinium salts and 2N-substituted 4(5)-aryl-2-amino-1H-imidazoles as inhibitors of biofilm formation by Salmonella Typhimurium and Pseudomonas aeruginosa. Bioorganic & Medicinal Chemistry 19(11), 3462-3473.
  • Steenackers, H., Ermolatev, D., Savaliya, B., De Weerdt, A., De Coster, D., Shah, A., De Vos, D., Vanderleyden, J., De Keersmaecker, S., Van der Eycken, E. (2011). Structure-Activity Relationship of 4(5)-Aryl-2-amino-1H-imidazoles, N1-Substituted 2-Aminoimidazoles and Imidazo[1,2-a]pyrimidinium Salts as Inhibitors of Biofilm Formation by Salmonella Typhimurium and Pseudomonas aeruginosa. Journal of Medicinal Chemistry, 54(2), 472-484.
  • Steenackers, H., Levin, J., Janssens, J., De Weerdt, A., Balzarini, J., Vanderleyden, J., De Vos, D., De Keersmaecker, S. (2010). Structure-activity relationship of brominated 3-alkyl-5-methylene-2(5H)-furanones and alkylmaleic anhydrides as inhibitors of Salmonella biofilm formation and quorum sensing regulated bioluminescence in Vibrio harveyi. Bioorganic & Medicinal Chemistry, 18(14), 5224-5233.
  • Ermolatev, D., Bariwal, J., Steenackers, H., De Keersmaecker, S., Van der Eycken, E. (2010). Concise and diversity-oriented route toward polysubstituted 2-aminoimidazole alkaloids and their analogues. Angewandte Chemie - International Edition, (49)49, 9465-9468.
  • Janssens, J., Steenackers, H., Robijns, S., Gellens, E., Levin, J., Zhao, H., Hermans, K., De Coster, D., Verhoeven, T., Marchal, K., Vanderleyden, J., De Vos, D., De Keersmaecker, S. (2008) Brominated furanones inhibit biofilm formation by Salmonella enterica serovar Typhimurium. Applied and Environmental Microbiology, 74(21), 6639-6648.
In silico screenings of anti-biofilm compounds
  • Qing, X., De Weerdt, A., De Maeyer, M., Steenackers, H.*, Voet, A.* (2017). Rational design of small molecules that modulate the transcriptional function of the response regulator PhoP. BBRC. Accepted.
  • Qing, X., Steenackers, H., Voet, A., De Maeyer, M. Computational studies of the active and inactive regulatory domains of response regulator PhoP using molecular dynamics simulations (2017). Molecular Informatics. doi: 10.1002/minf.201700031.
Mode of action determination of anti-biofilm compounds
  • Robijns, S., Roberfroid, S., Van Puyvelde, S., De Pauw, B., Uceda Santamaría, E., De Weerdt, A., De Coster, D., Vanderleyden, J., Steenackers, H. (2014). A GFP promoter fusion library for the study of Salmonella biofilm formation and the mode of action of biofilm inhibitors. Biofouling 30(5), 605-25.
Anti-biofilm coatings
  • Peeters, E., Hooyberghs, G., Robijns, S., De Weerdt, A., Kucharíková, S., Tournu, H., Braem, A., Čeh, K., Majdič, G., Španič, T., Pogorevc, E., Claes, B., Dovgan, B., Girandon, L., Impellizzeri, F., Erdtmann, M., Krona, A., Vleugels, J., Fröhlich, M., Garcia-Forgas, J., De Brucker, K., Cammue, BPA., Thevissen, K., Van Dijck, P., Vanderleyden, J., Van der Eycken, E., Steenackers, HP. (2018). An antibiofilm coating of 5-aryl-2-aminoimidazole covalently attached to a titanium surface. J Biomed Mater Res B Appl Biomater. 2018 Dec 13. doi: 10.1002/jbm.b.34283.
  • Claes, B., Boudewijns T., Muchez L., Hooyberghs G., Van der Eycken, E., Vanderleyden J., Steenackers H., De Vos D.E (2017). Smart metal-organic framework coatings: triggered anti-biofilm compound release. ACS Applied Materials & Interfaces, 9(5), 4440-4449.
  • Gerits, E., Kucharíková, S., Van Dijck, P., Erdtmann, M., Krona, A., Lövenklev, M., Fröhlich, M., Dovgan, B., Impellizzeri, F., Braem, A., Vleugels, J., Robijns, S.C., Steenackers, H.P., Vanderleyden, J., De Brucker, K., Thevissen, K., Cammue, B.P., Fauvart, M., Verstraeten, N., Michiels, J. (2016). Antibacterial activity of a new broad-spectrum antibiotic covalently bound to titanium surfaces. Journal of Orthopaedic Research, 34(12), 2191-2198.
Salmonella biofilms as a model system
  • Hermans, K., Roberfroid, S., Thijs, I.M., Kint, G., De Coster, D., Marchal, K., Vanderleyden, J., De Keersmaecker, S., Steenackers, H. (2015). FabR regulates Salmonella biofilm formation via its direct target FabB. BMC Genomics, 17, 253.
  • Van Assche, E., Van Puyvelde, S., Vanderleyden, J., Steenackers, H. (2015). RNA-binding proteins involved in post-transcriptional regulation in bacteria. Frontiers in Microbiology, 6:141.
  • Van Puyvelde, S., Steenackers, H.P., Vanderleyden, J. (2013). Small RNAs regulating biofilm formation and outer membrane homeostasis. RNA Biology, 10(2), 185-191.
  • Steenackers, H., Hermans, K., Vanderleyden, J., De Keersmaecker, S. (2012). Salmonella biofilms: An overview on occurrence, structure, regulation and eradication. Food Research International, 45, 502-531.

Screening platform

  • In vitro biofilm & toxicity
  • In silico: molecular modelling and datamining

Chemical synthesis

  • Novel synthesis pathways
  • Upscaling
  • Immobilization

Mode of action & resistance

  • GFP promoter fusions
  • Omics approaches
  • Experimental evolution

Towards application

  • Anti-biofilm coatings
  • Delivery (magnetic nanoparticles)
  • Combination therapy
  • Real life biofilms