Research at Costerton Biofilm Center

Biofilms are communities of aggregated bacterial cells embedded in an extracellular polymeric matrix, and they are associated with numerous chronic infections since bacteria in biofilms are tolerant to antimicrobial treatments and host immune defenses. Recent research suggests that the molecule cyclic diguanosine monophosphate (c-di-GMP) constitutes a ubiquitous intracellular regulator of bacterial biofilm formation. High levels of c-di-GMP induce formation of biofilms, whereas low levels of c-di-GMP induce a planktonic bacterial lifestyle. The level of c-di-GMP in bacteria is determined by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) which contain characteristic GGDEF and EAL/HD-GYP domains as well as regulatory domains. The research project is aimed at obtaining knowledge of the regulation of the activity of DGCs and PDEs, as well as on identification of down-stream c-di-GMP affecter proteins. This knowledge may reveal novel molecular targets for new drugs that can prevent or cure biofilm infections.

Contact

Tim Tolker-Nielsen
E-mail: ttn@sund.ku.dk
Tel: +45 26 24 56 90

Basically, bacteria display two life forms during growth and proliferation. In one form, the bacteria appear as single, independent cells (planktonic), and in the other form bacteria are organised in sessile aggregates. The latter form is commonly referred to as the biofilm phenotype. Acute infections are assumed to involve planktonic bacteria, and are generally treatable with antibiotics, though successful treatment depends on accurate and fast diagnosis. However, in cases where the bacteria succeed in forming a biofilm within the human host, the infection often turns out to be untreatable and will develop into a chronic state.

The important hallmark of chronic, biofilm-based infections is extreme resistance to antibiotics, as well as a number of other conventional antimicrobial agents and an extreme capacity to evade the host defense. In addition, since the bacteria in chronic infections are aggregated, resistance genes can be passed from one bacterium to the other.

We study the role of bacterial and fungal biofilms in chronic infections both in vitro, animal models and ex vivo material from chronic infections. Our interests are how bacteria initiate biofilms in the human body and why the immune defense seems to fail both in the initial infection and later in the chronic infection. What is the prevalence of bacteria and fungus on implants in general? What is the activity of the microbes in chronic infections and which species are present? We also seek to develop tools and methods to enable fast diagnosis of these infections, for better treatment and possible prevention.

The projects are in close collaboration with clinicians at most of the major hospitals in Denmark.

Projects focussed on these issues are currently funded by:
The Novo Nordisk Foundation Challenge program 2020, Persistent bacterial infections “PERFECTION”.

Contact

Thomas Bjarnsholt
E-mail: tbjarnsholt@sund.ku.dk
Tel: +45 20 65 98 88

The primary interest of the group is to cure persistent, chronic infections due to bacterial biofilms and the goal is to improve the treatment of this type of infections by interfering with the bacterial evolution, by enhancing the activity of antibiotics and by employing phage therapy.

1. Bacterial evolution and adaptation in biofilms
   Antimicrobial resistance (AMR) has become a significant and growing threat to public and environmental health and it has been identified as a major killer in our society. To face this problem a better understanding of the sources and mechanisms that contribute to the emergence and persistence of antibiotic resistance is required.
   Due to an elderly population and to an increase in the number of patients with life-style diseases, the number of patients with chronic, biofilm infections is on the rise. Antibiotic treatment can´t eradicate the biofilms due to their intrinsic tolerance (phenotypic resistance) and the development of mutational antibiotic resistance (heritable resistance) is accepted as a side –effect of the prolonged maintenance antibiotic therapy. In contrast to the planktonic, fast-dividing cells that are traditionally used to study antibiotic resistance development in shaking cultures, biofilm-grown bacteria are fundamentally different as they encounter gradients of nutrients and oxygen which lead to a heterogenous bacterial population including slow-growing or non-dividing cells. Distinct mutagenesis and selective processes have been described in non-dividing and nutrient-deprived bacterial cells and retromutagenesis involving oxidative lesions of the DNA have been shown to contribute to mutagenesis in ageing bacterial colonies. We have recently shown by in vitro experimental evolution studies of biofilms exposed to sub-inhibitory levels of ciprofloxacin that antibiotic resistance developed by distinct pathways in planktonic and biofilms, that oxidative stress plays an important role in this process and that besides development of resistance, bacteria adapt to antibiotic stress by rewiring their metabolic pathways.
   Purpose: to investigate the evolution and adaptation of bacteria in biofilms grown in in vivo-relevant microenvironments ( f. ex. anaerobiosis) and under exposure to antibiotics with different mode of action
   Understanding the bacterial evolution and adaptation during biofilm infections will elucidate the role of biofilms as a reservoir and persistence environments of AMR.
2. Control of pathogenic biofilms by bacteriophage-antibiotic combinations
   Antibiotic resistance has become a significant and growing threat to public and environmental health which triggered a renewed interest in bacteriophage therapy. As a last resort treatment of infections, phage therapy has successfully been used clinically on a compassionate basis as personalized therapy and in most of the cases in combination with antibiotics. Chronic infections caused by antibiotic tolerant biofilm-grown bacteria, are increasingly frequent and have been also identified as a target of phage therapy. We have recently shown that a combination of three lytic phages with ciprofloxacin was able to eradicate Pseudomonas aeruginosa PAO1 in vitro biofilm but repeated phage treatments enhanced the biofilm formation. Despite the increasing number of studies describing the biofilm eradication potential of treatment strategies involving phages, unintended induction of biofilm formation has also been described and this dual effect seems to depend on the specific bacterial strain-phage combination and the timing of phage addition. A better understanding of the phage-biofilm interactions is necessary for developing safe and predictable phage-based antimicrobial applications in the control of P. aeruginosa. The following two hypotheses are tested in this project: Hyp1: The outcome of phage/biofilm interactions depends on identifiable genetic factors in Pseudomonas aeruginosa; Hyp2: The control of pathogenic biofilms by phage/antibiotic therapies depends on the evolutionary pathways of bacterial defence against phages and/or antibiotics.
   To understand the role of the bacterial genomic diversity in the phage-biofilm interactions, especially the presence or absence in the genome of the CRISPR-Cas systems and of Pf-like filamentous profages, different P. aeruginosa clones with known genomic sequences will be investigated. Biofilms of each clone will be established in different biofilm systems and will be exposed to lytic phages and to anti-pseudomonal antibiotics. The outcome of the phage/biofilm interactions will be studied by monitoring the biofilm structures in confocal-laser scanning microscope. The temporal evolution of resistance to phage and antibiotics will be investigated in the harvested biofilm populations during an experimental evolution study in MBEC assay.
   Unraveling the complex interaction between biofilm bacterial populations, phages and different classes of antibiotics on several P. aeruginosa bacterial clones will allow qualified predictions of efficient phage-antibiotic combination with minimal phage and antibiotic resistance development, suitable for treatment of chronic infections.

Funding:

KU, DTU, Læge Sofus Carl Emil Friis og Hustru Olga Doris Friis' Legat, Aase og Ejnar Danielsens Fond, Danish Cystic Fibrosis Association, Frøken P-A. Brandt, Gunna and Einer Polanders Legat

Collaborations:

• Copenhagen Cystic Fibrosis Department, Rigshospitalet, Copenhagen, Denmark
• DTU Biosustain, Novo Nordisk Foundation Center for Biosustainability, Denmark
• DTU Bioengineering, Institut for Bioteknologi og Biomedicin, Denmark
• KU, Marine Biological Section, Denmark
• University of Leiden, Division of Systems Biomedicine and Pharmacology, the Leiden Academic Centre for Drug Research (LACDR), The Netherlands
• Helmholtz Centre for Infection Research, Hannover, Germany
• Universitat des les Illes Balears, Palma de Mallorca, Spain

Contact

Oana Ciofu
E-mail: ociofu@sund.ku.dk
Tel: + 45 29 21 73 67

Biofilms formed by opportunistic pathogenic bacteria can resist immune defenses and antimicrobial therapy, and therefore cause considerable problems in medical settings. However, if forced away from the protective biofilm-state to a free-living planktonic mode, the bacterial cells become susceptible to the action of the immune system and antimicrobials. Current research indicates that the secondary messenger c-di-GMP is a general controller of the biofilm lifestyle in bacteria. High internal levels of c-di-GMP induce production of extracellular matrix components which promote biofilm formation, whereas low c-di-GMP levels down-regulate the production of extracellular matrix components and lead bacteria to the planktonic mode of life. We use a high-throughput screening approach for identification of compounds that can reduce the c-di-GMP content in bacteria. Such compounds are expected to serve as lead molecules for the development of drugs which can interfere with biofilm formation, and cure problematic bacterial infections.

Projects focused on these issues are funded by:

The Danish Council for Independent Research, the Lundbeck Foundation, and the Novo Nordisk Foundation.

Contact

Tim Tolker-Nielsen
E-mail: ttn@sund.ku.dk
Tel: +45 26 24 56 90

Michael Givskov
E-mail: mgivskov@sund.ku.dk
Tel: +45 21 40 98 67

It is well established that bacterial biofilms are more tolerant to antibiotics than planktonic bacteria. Several factors are known to play a role in the high antimicrobial tolerance displayed by biofilms, including restricted antibiotic penetration, physiological heterogeneity, and induction of specific antibiotic tolerance genes. However, more knowledge is warranted about antibiotic tolerance mechanisms in order for us to develop effective treatments against biofilm infections. This project focuses on obtaining knowledge about the role of the extracellular matrix, differential growth activity, and heterogenic gene expression in biofilm-associated antimicrobial tolerance.

Projects focused on these issues are funded by:

The Danish Council for Independent Research.

Contact

Tim Tolker-Nielsen
E-mail: ttn@sund.ku.dk
Tel: +45 26 24 56 90

The Danish National Institute of Public Health has implemented the population study “Danish Health Examination Survey with Focus on Lifestyle and General Health”. To this study is linked an odontological examination called The Oral Health Study.

The main purposes of this study are:

1. to survey on incidence of dental diseases and oral health habits in a Danish adult population and
2. to analyze the association between oral health, in particular the dental biofilm-related diseases caries and periodontitis, and general health, including effects of lifestyle and medication. Important lifestyle factors are: nutrition, alcohol consumption, smoking, and exercise. Saliva samples are subjected to microarray-based identification of the oral bacterial species. The bacterial species profiles are related to dental diseases and the lifestyle factors.

One of the most important characteristics of biofilms is that the bacteria in these sessile microbial communities display a remarkable increased tolerance to antimicrobial attack. Because of their innate resistance to host immune systems, antibiotics, and biocides, biofilms are difficult, if not impossible, to eradicate. Biofilm formation therefore leads to various persistent infections in humans and animals.

Because the use of conventional antimicrobial compounds in many cases cannot eradicate biofilms, there is an urgent need to develop new ways to prevent biofilm formation or to remove existing biofilm. We have initiated a research project aimed at obtaining knowledge about the molecular mechanisms which underlay tolerance to antimicrobial agents in oral biofilms. Such knowledge is necessary in order to develop compounds which can interfere with tolerance development, and thus make the biofilms susceptible to conventional antimicrobial compounds.

Projects focussed on these issues are currently funded by: Tandlægefonden.

Contact

Michael Givskov
E-mail: mgivskov@sund.ku.dk
Tel: +45 21 40 98 67

Tim Tolker-Nielsen
E-mail: ttn@sund.ku.dk
Tel: +45 26 24 56 90

Daniel Belstrøm
E-mail: dbel@sund.ku.dk

Bacterial biofilms may cause chronic infections due to their ability to evade clearance by the immune system and antibiotics. The persistent biofilms induce a hyper inflammatory state that damages the surrounding host tissue. Knowledge about the components of biofilms that are responsible for provoking the harmful but inefficient immune response is limited. In this research project we study the role of extracellular biofilm matrix polysaccharides in activation of polymorphonuclear leukocyte (PMN) responses towards biofilms.

Projects focused on these issues are funded by:

The Danish Council for Independent Research.

Contact

Tim Tolker-Nielsen
E-mail: ttn@sund.ku.dk
Tel: +45 26 24 56 90

Atopic dermatitis (AD) is a common, chronic skin condition associated with allergies, itching, sleep disruption and reduced quality of life. Certain bacteria, e.g. Staphylococcus, exhibit increased growth on the skin of AD patients compared to healthy controls. It is unknown whether changes in skin bacteria composition (the so-called “microbiome”) precede eczema, or vice versa. Staphylococci, particularly, increase in numbers during eczema exacerbation and cause serious infections, but repeated use of antibiotics may lead to bacterial resistance. Consequently, it is difficult for clinicians to properly administer disinfectants and antibiotics to prevent AD and flare-ups. Our projects will follow large groups of healthy adults and newborns and adult patients with AD to determine the role of skin bacteria in AD. The findings will help clinicians identify the correct treatment strategies to prevent and treat AD, as well as assist drug developers to develop improved patient solutions.

Funded by:
The LEO Foundation: The spatial composition and distribution of the cutaneous microbiota in atopic dermatitis and healthy skin.

The Novo Nordisk Foundation Tandem Program 2019: The temporal and physiological importance of inflammation and bacteria in atopic dermatitis, in Collaboration with professor Jacob Thyssen, Gentofte Hospital

Contact

Thomas Bjarnsholt
E-mail: tbjarnsholt@sund.ku.dk
Tel: +45 20 65 98 88