Which individual human factors favour the formation of biofilms?

What is this research project about?

Scanning electronic image of a macrophage (red) and the bacterium Pseudomonas aeruginosa (blue). © Prof. Manfred Rohde, Dr. Sebastian Felgner

What is this research project about?

Implant-associated infections represent a serious health burden in the clinics. They are the consequence of microorganisms that are able to colonise biological surfaces or surfaces of indwelling medical devices, and to form biofilms there. Biofilms are communities of microorganisms attached to such hydrated interfaces and enclosed in self-produced extracellular matrices. These structures present a considerable therapeutic challenge. Bacteria in biofilms exhibit altered phenotypes in their growth rates, gene expression profiles, and protein production. Embedded in their extracellular matrix, they become resilient to both antibiotics and the effector mechanisms of the immune system of the host. Since antibiotics treatment alone is apparently ineffective to treat these infections, the only possible treatment is the removal of the infected device followed by antibiotics. All of which results in high costs for both the patients and the healthcare facilities.

Made visible with immunofluorescence staining: cell nuclei (blue), macrophages (red)

and Pseudomonas aerugina (green). © Dr. Sebastian Felgner

What’s the current status?

The connections between the formation of bacterial biofilms on implants and the immune system of the host have not been investigated in detail. Thus, still little is known on the role of the immune system in the development of implant-associated biofilms produced by S. aureus or P. aeruginosa or other pathogenic bacteria. However, for rational design of treatment strategies, this would be an essential asset.

To develop a mouse model for studying implant-associated infections, we pre-colonized osmotic pumps with S. aureus and implanted them subcutaneously into C57Bl/6 mice. The bacteria expressed a bacterial luciferase which allowed monitoring of their expansion and the development of biofilms by employing noninvasive in vivo imaging. Subsequently, the presence of bacterial aggregates and biofilms on the surface of the implants was showed by immunofluorescence and electron microscopy. Additionally, myeloid cells, including neutrophils and monocytes, had been attracted to the colonized pumps and formed a barrier for the bacteria towards the tissue surrounding the implant. Despite the close neighborhood of bacteria and myeloid cells, no phagocytosis had taken place. Only containment of bacteria to the pumps could be observed. However, activation of myeloid cells had taken place. Depletion of neutrophils led to an enhanced infection and to dissemination of the bacteria from the pumps to the neighboring tissue.

Under the microscope: skin samples with Staphylococcus

aureus within cellular traps (arrows).

Immunofluorescence staining: Pseudomonas aeruginosa bacteria whose flagella are stained green. © Dr. Sebastian Felgner

What are the project goals?

Our previous experiments using osmotic pumps loaded with immune modulating agents demonstrated that biofilm formation by bacterial pathogens like S. aureus can be influenced by manipulating the myeloid cells attracted to the colonized devices. First, these findings should be expanded to other biofilm forming bacterial pathogens. Therefore, the general objective of this proposal is to characterize the attracted myeloid cells as well as the biofilm residing bacteria in great detail. The phenotype of such myeloid cells, which is most likely of inflammatory type, should then be modulated using the osmotic pumps as source of exogenously supplied agents. Similarly, deletion mutants of the bacteria as well as recombinant murine hosts should be employed to further acquire information on the biofilm residing bacteria and the possibility of modulation.

How do we get there?

Osmotic pumps will be exposed to bacterial cultures in order to pre-colonize the devices. The pumps will then be surgically administered subcutaneously to the mice and monitored for various time periods. A major technique to quantitate the bacteria in vivo will consist of non-invasive imaging of the bacteria. Thus, bacteria will be used that express the bacterial luciferase and can be detected within the living mouse using the IVIS systems. Plating under these circumstances will not provide valid data since we have noticed that part of the bacteria might be lost when the pumps are removed from the animals. On the other hand, non-invasive imaging allows following expansion or reduction of the number of bacteria on the device in real time and data can be acquired from the same individual mouse over time. The IVIS will also be employed to quantitate the bacteria under antibiotic therapy. The bacteria should be resilient against treatment since a mature biofilm should be protective. Modulated biofilms might be less protective. This would provide a simple read out system for our experiments. Analysis will also employ microscopic examination of the colonized pumps using light sheet a well as scanning electron microscopy. The latter will provide high resolution pictures while for light sheet microscopy biofilms can be stained using fluorescent antibodies against components of the biofilm or against the bacteria.

Under the microscope: surface of osmotic pumps, the anti-S. aureus antibodies are shown in green.

Scanning electron microscopy of colonized implant surfaces.

Images modified by https://doi.org/10.3389/fimmu.2019.01082 © by the authors.

Mouse model for implant-associated infections.


Project title: Identifying host factors that drive biofilm formation

Prof. Dr. Reinhold Förster

Projekte: B5, B9, C2

Prof. Dr. Susanne Häußler

Projekte: C1, C2

Prof. Dr. Meike Stiesch

Projekte: C1, C2

Project C2 Publications

Publications 2022

High plasmidome diversity of extended-spectrum beta-lactam-resistant Escherichia coli isolates collected during one year in one community hospital. Neffe L, Abendroth L, Bautsch W, Häussler S, Tomasch J. Genomics. 2022 Apr 18:110368.

Antibacterial and Cytocompatible: Combining Silver Nitrate with Strontium Acetate Increases the Therapeutic Window. Parizi MK, Doll K, Rahim MI, Mikolai C, Winkel A, Stiesch M.  Int J Mol Sci. 2022 Jul 22;23(15):8058. doi: 10.3390/ijms23158058. PMID: 35897634; PMCID: PMC9331456.

The membrane-active polyaminoisoprenyl compound NV716 re-sensitizes Pseudomonas aeruginosa to antibiotics and reduces bacterial virulence. Wang G, Brunel JM, Preusse M, Mozaheb N, Willger SD, Larrouy-Maumus G, Baatsen P, Häussler S, Bolla JM, Van Bambeke F. Commun Biol. 2022 Aug 25;5(1):871. doi: 10.1038/s42003-022-03836-5. PMID: 36008485; PMCID: PMC9411590.

Publications 2021

The effect of keratinized mucosa on the severity of peri-implant mucositis differs between periodontally healthy subjects and the general population: a cross-sectional study. Kabir L, Stiesch M, Grischke J.  Clin Oral Investig. 2021 Mar;25(3):1183-1193. doi: 10.1007/s00784-020-03422-1. Epub 2020 Jul 1. PMID: 32607828; PMCID: PMC7878216.

The human oral phageome. Szafrański SP, Slots J, Stiesch M.  Periodontol 2000. 2021 Jun;86(1):79-96. doi: 10.1111/prd.12363. Epub 2021 Mar 10. PMID: 33690937.

Publications 2020

Early host-microbe interaction in a peri-implant oral mucosa-biofilm model. Mikolai C, Kommerein N, Ingendoh-Tsakmakidis A, Winkel A, Falk CS, Stiesch M. Cell Microbiol. 2020 Aug;22(8):e13209. doi: 10.1111/cmi.13209. Epub 2020 May 14. PMID: 32329166.

Evidence for inoculum size and gas interfaces as critical factors in bacterial biofilm formation on magnesium implants in an animal model. Rahim MI, Szafrański SP, Ingendoh-Tsakmakidis A, Stiesch M, Mueller PP. Colloids Surf B Biointerfaces. 2020 Feb;186:110684. doi: 10.1016/j.colsurfb.2019.110684. Epub 2019 Nov 28. PMID: 31812076.

Publications 2019

IL-1β Promotes Staphylococcus aureus Biofilms on Implants in vivo. Gutierrez Jauregui R, Fleige H, Bubke A, Rohde M, Weiss S, Förster R. Front Immunol. 2019 May 17;10:1082. eCollection 2019.

Establishment of an induced memory response in Pseudomonas aeruginosa during infection of a eukaryotic host. Kordes A, Grahl N, Koska M, Preusse M, Arce-Rodriguez A, Abraham WR, Kaever V, Häussler S. ISME J. 2019 Aug;13(8):2018-2030. doi: 10.1038/s41396-019-0412-1. Epub 2019 Apr 5. PMID: 30952997; PMCID: PMC6775985.

Keratinized mucosa width is associated with severity of peri-implant mucositis. A cross-sectional study. Jasmin Grischke, Annika Karch, Andreas Wenzlaff, Magdalena Marta Foitzik, Meike Stiesch, Jörg Eberhard. Clinical Oral Implants Research. First published: 10 April 2019. https://doi.org/10.1111/clr.13432

Genetically diverse Pseudomonas aeruginosa populations display similar transcriptomic profiles in a cystic fibrosis explanted lung. Kordes A, Preusse M, Willger SD, Braubach P, Jonigk D, Haverich A, Warnecke G, Häussler S.  Nat Commun. 2019 Jul 30;10(1):3397. doi: 10.1038/s41467-019-11414-3. PMID: 31363089; PMCID: PMC6667473.

Publications of the Project C2