biofilms 9.3

For decades, biofilm research has been trying to provide a comprehensive description of the biofilm matrix. Due to the complexity of the biofilm matrix, an easy characterisation seems somehow impossible. Recently, optical coherence tomography has been pushed and very much contributes to an advanced structural characterisation of the mesoscopic biofilm structure. However, the biofilm matrix is a highly complex matter with various physical (e.g. distribution of biomass, material properties), chemical (e.g. composition, constituents) and (micro)biological (e.g. microorganisms and interactions) properties.
We are thus looking for contributions improving the structural (physical, chemical, micro biological) understanding of the biofilm matrix. Very much appreciated are inputs correlating results of different methodologies.

Chair: Per Halkjær Nielsen | Co-chair: Harald Horn
| Wed, 30 Sep, 12:10–15:30
| Attendance Wed, 30 Sep, 16:40–18:10

Topic assets

Attendance time: Wednesday, 30 September 2020, 16:40–18:10

Cristina D. Cruz, Rebekah C. Hewitt, and Päivi Tammela

Antibiotic resistance is expected to cause 10 million deaths per year worldwide by 2050. One of the mechanisms for the resilient nature of bacteria toward antibiotics is through the formation of biofilm. Bacterial biofilms are sessile communities of microorganisms, which exist in a matrix of proteins, carbohydrates, eDNA and other various components – collectively known as extracellular polymeric substances. Biofilms slow the penetration of drugs, and also contribute to the development of a resistant phenotype known as persisters. Thus, understanding biofilm composition might contribute to the development of anti-biofilm strategies. The aim of this study was to explore biofilm formed by five Staphylococcus spp ATCC strains, commonly used in research as references: S. aureus 25923, S. aureus 29213, S. aureus 43300 (methicillin-resistant), S. aureus 6538 and S. epidermidis 12228. Biofilm mass and its components were analysed after 24h and 72h of biofilm growth. Bacterial biofilm was prepared in 96-well microtiter plates, in Trypticase Soy Broth supplemented with 1% glucose. After incubation at 37°C, absorbance measurements and crystal violet staining were performed and the specific biofilm formation determined for each strain. Extracellular polymeric substances were extracted using a combination of physical and chemical methods; including centrifugation, vortexing and the use of 1.5M NaCl. In these assays, biofilms were grown in polystyrene tubes containing 10 ml of same media mentioned above. The concentration of protein, carbohydrate and eDNA was determined using the Bicinchoninic acid assay, phenol-sulfuric acid method and DNeasy® Blood and Tissue Kit, respectively, followed by spectroscopy. Our data demonstrated heterogeneity between the biofilm-forming capabilities and EPS components within staphylococcal strains and species. Strains 25923 and 6538 had the highest value for biofilm formation at both time points. Interestingly, strain 43300 was the only one to show a significant increase in biofilm after 72h. Contradictory to previous findings, S. epidermidis 12228 was found to be a good biofilm producer. At both time points studied, strains demonstrated considerably higher concentrations of protein (varying from 172 µg/mL – 345 µg/mL) and carbohydrate (56 µg/mL - 372µg/mL) in EPS compared to eDNA (2.74 µg/mL – 8.12 µg/mL). On average, strains 43300 and 12228 had the highest concentration of protein, and the latter also had the highest carbohydrate and eDNa amounts at 72h. Strains 25923 and 6538 had a significant decrease in eDNA concentration over time. Based on this brief study, the relative quantities of EPS components investigated is similar to that of other studies with protein being the most plentiful component followed by carbohydrate and then considerably lower amounts of eDNA. Differences in specific biofilm formation did not directly reflect variations observed in abundance of a particular constituent in the matrix of EPS. This study also showed that S. epidermidis 12228, usually classified as a weak or non-biofilm former, was able to grow a relatively substantial biofilm under the conditions tested here.

How to cite: D. Cruz, C., C. Hewitt, R., and Tammela, P.: A brief exploration of EPS composition in biofilms of Staphylococcus spp ATCC reference strains, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-129,, 2020.

Giovanni Savorana, Roberto Rusconi, Roman Stocker, and Eleonora Secchi

In many environmental or medical settings, biofilm formation is the most successful strategy for bacterial colonization1,2. In the biofilm lifestyle, bacteria embed themselves in a self-secreted matrix of extracellular polymeric substances (EPS), acting as a shield against mechanical and chemical insults3. When ambient flow is present, this viscoelastic EPS scaffold can take a streamlined shape, forming biofilm threads suspended in flow, called streamers4. In many situations, the streamer architecture can enhance the harmful effects of biofilms, bridging the spaces between obstacles in the flow path5. Despite their importance for biofilm survival, little is known about the material properties of the matrix. In particular, these are really hard to probe with traditional rheological techniques when the biofilm grows into the thread-like streamer shape.

In this work we present a microfluidic platform that allows to reproducibly grow biofilm streamers in controlled chemical and flow conditions and to characterize their structure and mechanical properties in situ6.This platform overcomes the main sources of error and variability of the experiments performed with traditional flow-chambers: the randomness in the location and shape of the streamers. Our device consists of a straight channel with isolated micropillars, where a bacterial suspension is injected at a constant flow rate. The micropillars act as nucleation points for the growth of a pair of biofilm filaments, developing on the midplane of the channel under the action of secondary flows. The microfluidic technology allows to control the chemical and flow conditions and to perform live imaging of the growth process. By controlling the flow rate, we are also able to perform in situ stress tests on the streamers by inducing controlled variations of the fluid shear stress exerted on them. We developed a theoretical framework to estimate the material properties of biofilm streamers from the flow-induced deformation measured in our experiment. Thanks to this platform, we are able to investigate the role of the different EPS components7and the physico-chemical microenvironment in determining biofilm structure and rheology.


1Flemming and Wingender, Nat. Rev. Microbiol. 8, 623 (2010).

2Flemming et al., Nat. Rev. Microbiol. 14, 563 (2016).

3Peterson et al., FEMS Microbiol. Rev. 39, 2 (2015).

4Rusconi et al., J. R. Soc. Interface 7, 1293 (2010).

5Drescher et al., Proc. Natl. Acad. Sci. 112, 11353 (2015).

6Savorana et al., paper in preparation

7Secchi et al., paper in preparation

How to cite: Savorana, G., Rusconi, R., Stocker, R., and Secchi, E.: A microfluidic platform for characterizing the structure and rheology of biofilm streamers, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-76,, 2020.

A particle-based model to investigate nanoparticle diffusion in a 3D biofilm environment
Bart Coppens, Jiří Pešek, Bart Smeets, and Herman Ramon
Laura Kuschmierz, Martin Meyer, Benjamin Meyer, Sonja-Verena Albers, Christopher Bräsen, Jost Wingender, Oliver J. Schmitz, and Bettina Siebers

Archaea, representatives of the third domain of life, are often referred to as “extremophiles” since most of the cultivable species are adapted to extreme environments [1]. However, environmental cultivation-independent approaches (metagenomics) revealed a wide distribution of Archaea in moderate habitats suggesting a major role in geochemical processes. Similar to Bacteria, also Archaea are believed to exist predominantly in the biofilm mode, but knowledge about archaeal biofilm formation and structure, extracellular polymeric substance (EPS) composition and synthesis is scarce [2].

Sulfolobus acidocaldarius is a thermoacidophilic, aerobic Crenarchaeon (78°C and pH 2-3) that was isolated from acid hot springs [3]. The organism is easy to cultivate under laboratory conditions and a genetic system is established. In this study, we investigate S. acidocaldarius biofilms with a special focus on synthesis and transport of exopolysaccharides (PS). PS constitute a major EPS component beside proteins and eDNA, suggesting an important role in Sulfolobus biofilms, and changes in PS composition were observed in response to environmental stress [4]. A gene cluster encoding several glycosyltransferases (GTs) as well as membrane proteins (MPs), likely involved in exopolysaccharide synthesis, was identified in S. acidocaldarius. Several deletion mutants have been constructed lacking certain GT and MP encoding genes from the PS gene cluster. A combination of methods including the quantification of biofilm formation, isolation and quantification of EPS components, visualization of biofilm and PS structures via confocal laser scanning microscopy as well as molecular and biochemical techniques have been applied to compare biofilm characteristics of wildtype and mutant strains. First insight into the function of GTs and MPs will be presented and a model of PS synthesis and export will be proposed.

[1] Schocke et al. (2019). Curr. Opin. in Biotechnol. 59, 71-77.

[2] van Wolferen et al. (2018). Nature Rev. Microbiol. 16(11), 699-713.

[3] Brock et al. (1972). Arch. Mikrobiol. 84, 54-68.

[4] Jachlewski et al. (2015). Front. Bioeng. Biotechnol. 3, 123.


How to cite: Kuschmierz, L., Meyer, M., Meyer, B., Albers, S.-V., Bräsen, C., Wingender, J., Schmitz, O. J., and Siebers, B.: Archaeal biofilms: Composition of extracellular polymeric substances, exopolysaccharide synthesis and secretion in Sulfolobus acidocaldarius, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-59,, 2020.

Sarah Zecchin, Nicoletta Guerrieri, Evelien Jongepier, Leonardo Scaglioni, Gigliola Borgonovo, Gerard Muyzer, and Lucia Cavalca

Arsenic is a toxic but naturally abundant metalloid that globally leads to contamination in groundwater and soil, exposing millions of people to cancer and other arsenic-related diseases. In several areas in Northern Italy arsenic in soil and water exceeds law limits (20 mg kg-1 and 10 mg L-1, respectively), due to both the mineralogy of bedrock and former mining activities. The Rio Rosso stream, located in the Anzasca Valley (Piedmont) is heavily affected by an acid mine drainage originated from an abandoned gold mine. Arsenic, together with other heavy metals, is transferred by the stream to the surrounding area. The stream is characterized by the presence of an extensive reddish epilithic biofilm at the opening of the mine and on the whole contaminated waterbed.

The aim of this study was to characterize the mechanisms allowing the biotic fraction of this biofilm to cope with extreme arsenic concentrations. The composition and functionality of the microbial communities constituting the epilithic biofilms sampled in the close proximity and downstream the mine were unraveled by 16S rRNA genes and shotgun Illumina sequencing in relation to the extreme physico-chemical parameters. In parallel, autotrophic and heterotrophic microbial populations were characterized in vivo by enrichment cultivation and isolated strains were tested for their ability to perform arsenic redox transformation.

Preliminary analyses indicated that the biofilm accumulated arsenic in the order of 6 · 103 mg kg-1, in contrast to 0.14 mg L-1, measured in the surrounding water. The main chemical parameter affecting the composition of the microbial community was the pH, being 2 next to the mine and 6.7 in the downstream sampling point. In both sampling sites iron- and sulfur-cycling microorganisms were retrieved by both cultivation and molecular methods. However, the diversity of the microbial community living next to the mine was significantly lower with respect to the community developed downstream. In the latter, autotrophic Cyanobacteria belonging to the species Tychonema were the dominant taxa. A complete arsenic cycle was shown to occur, with heterotrophic bacteria mainly responsible for arsenate reduction and autotrophic bacteria performing arsenite  oxidation.

These observations indicate that the epilithic biofilm living in the Rio Rosso stream represents a peculiar ecosystem where microorganisms cope with metalloid toxicity likely using diverse mechanisms. Such microbial metabolic properties might be exploited in bioremediation strategies applied in arsenic-contaminated environments.

How to cite: Zecchin, S., Guerrieri, N., Jongepier, E., Scaglioni, L., Borgonovo, G., Muyzer, G., and Cavalca, L.: Assessing arsenic bioremediation potential of epilithic biofilms affected by acid-mine drainage, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-81,, 2020.

Iztok Dogsa, Rok Kostanjsek, and David Stopar

Planktonic bacterial cells are by definition not aggregated. However, our previous work, where we have demonstrated the invisible mechanical connections between bacterial cells in dilute planktonic suspensions, challenged this assumption. Here we provide an experimental evidence using autocorrelation analysis of micrographs that in planktonic suspensions of B. subtilis a size continuum of aggregated structures is formed. In the microbial aggregates viable cells were embedded in the nucleic acid network. The eDNA was released during regular cell lysis events. To determine the size distribution of planktonic bacterial aggregates a pair-wise spatial correlations of bacterial cells in microscopic images were calculated. The monotonously decreasing shape of the autocorrelation function indicated a continuous distribution of bacterial aggregate sizes from monomer to multimers. Soft bacterial aggregates in dilute suspensions provide a missing link in a continuum of organic matter in aqueous environments and can significantly improve our understanding how non-attached biofilms form during planktonic growth.

How to cite: Dogsa, I., Kostanjsek, R., and Stopar, D.: A thin line between plankton and biofilm, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-157,, 2020.

Bacterial Differentiation in Oral Multi-Species Biofilm Models Using Confocal Raman Microscopy
Lukas Kriem, Steffen Rupp, Kevin Wright, and Renzo Ccahuana-Vasquez
Francisco Diaz-Pascual and Knut Drescher

Bacterial cells are often exposed to stress by changes in their environment. During the last decades the response of isolated cells to stress has been investigated in great detail. By contrast, little is known about the emergent multicellular level responses to stress, such as antibiotic exposure. Studying responses at the community level is key to understand the structure and function of the most common bacterial state: the multicellular communities termed biofilms. Here, by analysing Vibrio cholerae biofilms exposed to all different classes of antibiotics with single-cell resolution, we found that inhibition of protein synthesis cause striking changes in cell volume and biofilm architecture. The observed changes in cell volume are a single-cell level response driven by metabolic effects of the translational inhibition. The multicellular-level responses result from changes in matrix composition, matrix-cell dissociation and mechanical properties of the biofilms. We observed that these antibiotic-induced changes in biofilm architecture have strong consequences on the ecological dynamics of biofilms by making biofilms prone to invasion by bacteriophages and other bacterial cells. These mechanistic and ecological consequences of the emergent group-level architectural response to antibiotics are important to fully understand the ecological succession of biofilms and the implications of antibiotic therapy.

How to cite: Diaz-Pascual, F. and Drescher, K.: Biofilm Architectural Breakdown in Response to Antibiotics Facilitates Community Invasion, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-21,, 2020.

Piet Lens

Feeding cadmium (II) and selenium (IV) simultaneously to anaerobic granular sludge with the aim to synthesize cadmium selenide (CdSe) nanoparticles induces compositional changes in the extracellular polymeric substances (EPS) matrix of this sludge. A methanogenic anaerobic granular sludge was repeatedly exposed to Cd(II) (10-50 mg L-1) and selenite (79 mg L-1) for 300 days at pH 7.3 and 30 °C in a fed-batch feeding regime for enrichment of Se reducing bacteria and synthesis of CdSe nanoparticles. EPS fingerprints of the granular sludge, obtained by size exclusion chromatography coupled to a fluorescence detector, showed a significant increase in the intensity of protein-like substances with >100 kDa apparent molecular weight (aMW) upon repeated exposure to Cd(II) and Se(VI). This was accompanied by a prominent decrease in protein-like substances of aMW <10 kDa. The fingerprint of the humic-like substances showed emergence of a new peak with aMW of 13 to 300 kDa in the EPS extracted from the Cd/Se fed granular sludge. Experiments on metal(loid)–EPS interactions showed that the CdSe nanoparticles interact mainly with loosely bound-EPS (LB-EPS). This study showed that the formation of Se(0) and CdSe nanoparticles occurs in the LB-EPS fraction of the granular sludge and repeated exposure to Cd and Se induces compositional changes in the EPS matrix.

How to cite: Lens, P.: Cadmium selenide formation influences the production and characteristics of extracellular polymeric substances of anaerobic granular sludge, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-60,, 2020.

Martin Meyer, Laura Kuschmierz, Bettina Siebers, Jost Wingender, and Oliver J. Schmitz

Microorganisms, such as archaea, favour life in a biofilm rather than the planktonic form of life. A biofilm is defined as a community of microorganisms embedded in a self-produced matrix of hydrated extracellular polymeric substances (EPS), mainly polysaccharides (PS), proteins and extracellular DNA. The polysaccharides form a three-dimensional network, which provides stability of the biofilm and mediates the adhesion to surfaces. [1] Analysis of the monomeric composition of PS requires chromatographic separation and identification by mass spectrometry (MS).

A comparative study of different chromatographic methods for the analysis of the monomeric composition of exopolysaccharides from archaeal biofilms from Sulfolobus acidocaldarius has been carried out. For this study, different chromatographic separation methods, such as supercritical fluid chromatography (SFC), hydrophilic interaction liquid chromatography (HILIC) reversed-phase liquid chromatography (RP-LC) and gas chromatography (GC), each coupled to mass spectrometry, were developed and compared by means of separation performance and sensitivity, using authentic standards.

The study revealed, that each method features distinct advantages and disadvantages over the other methods. For example, when using SFC-MS, no derivatization is necessary and soft ionization conditions can be used. [2] However, the HILIC-MS and RP-LC-MS methods show significantly greater separation performances for the analysis of the monosaccharide composition. [3] All investigated methods show similar quantification limits in the sub-mg/L range.

Finally, the developed chromatographic methods were applied to real biofilm samples of the thermoacidophilic archaeon Sulfolobus acidocaldarius. To determine the monomeric composition of the exopolysaccharides from these archaeal biofilms, the extracellular polymeric substances were extracted from the biofilm and then the PS were hydrolyzed.



[1] H.-C. Flemming, Nat. Microbiol. Rev. 2010, 8, 623 - 633. [2] M. Lafosse, J. Chromatogr. A, 1996, 720, 61-73. [3] V. Sieber, J. Chromatogr. A, 2014, 1350, 44–50.

How to cite: Meyer, M., Kuschmierz, L., Siebers, B., Wingender, J., and Schmitz, O. J.: Comparative study of chromatographic methods for the analysis of exopolysaccharides from archaeal biofilms, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-58,, 2020.

Cristina Amador, Henriette L. Røder, Ute Kuhlicke, Thomas Neu, and Mette Burmølle

The biofilm matrix contributes to the establishment of microbial cells on very diverse surfaces, stabilizing biofilms and providing cells with protection against multiple hostile conditions. Moreover, the biofilm matrix can also retain nutrients, enzymes or quorum sensing molecules, favoring the establishment of social interactions among biofilm cells. Functional bacterial amyloids are part of the biofilm structural components of various species, and they were previously proven to bind QS molecules and strengthen the matrix. Multiple studies have been conducted to characterize matrix determinants and their regulation in single species biofilms, while these remain scarcely understood in multispecies biofilms. We have previously isolated and characterized a soil-derived consortium composed of Xanthomonas retroflexus, Stenotrophomonas rhizophila, Microbacterium oxydans and Paenibacillus amylolyticus showing enhanced biofilm biomass and differential gene/protein expression specific of the four-species biofilm.

This study aimed at exploring the effect of interspecies interactions on biofilm matrix production in the four-species biofilm. We hypothesize that interspecies interactions may result in differential expression of matrix-encoding genes responsible for biofilm emergent properties.

We searched for matrix determinant homologues in X.retroflexus and combined different techniques for characterizing the matrix identity and expression in mono-, dual- and multispecies biofilms.

The fap amyloid operon, described in Pseudomonas as a biofilm-scaffold contributing element, was deleted in X. retroflexus, replaced in the four-species model and compared to the parental community for biofilm structure and adhesion capability. The fap mutant displayed poor substrate colonization in flow cells in both mono- and multispecies biofilms with relative filamentous structure compared to the parental strain/ consortium. However, adhesion did not significantly change under static conditions. To characterize matrix composition, we tested 78 different lectins in multispecies biofilms and identified five that bound to our samples. Interestingly, some matrix glycoconjugates were only produced in the consortium.

Our data suggest that loss of matrix components, such as the Fap amyloid, and the presence of other species, influences synergistic biofilm properties in the four-species consortium. Ongoing approaches involving localized expression of matrix-encoding genes and matrix proteomes will aid in identifying the mechanisms underlying emergent properties in the four-species biofilm.


How to cite: Amador, C., L. Røder, H., Kuhlicke, U., Neu, T., and Burmølle, M.: Decrypting the matrix: how interspecies interactions influence matrix in multispecies biofilm?, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-98,, 2020.

Extracellular DNA and Outer Membrane Vesicles contribute to P. fluorescens SBW25 air-liquid interface biofilms.
Olena Moshynets, Airat Kayumov, Olga Iungin, Svitlana Rymar, Ianina Pokholenko, and Andrew Spiers
Carla Perez-Cruz, Monica Estupiñan, Mª Goretti Llamas-Arriba, Oier Etxebeste, Anders Lanzen, and Laura Alonso-Saez

Alteromonas are model copiotrophic marine bacteria that are able to produce highly hydrated extracellular biopolymers mainly composed of polysaccharides (i.e., extracellular polysaccharides, EPS), which have a role in biofilm formation in oceans. Some of the functions of EPS are related to protection against environmental stressors, adhesion to particles, carbon storage and nutrient acquisition. Microbial EPS are largely heterogeneous in composition and structure, and some strains produce different types of EPS in response to different conditions. This study aimed at characterizing the synthesis of polysaccharides secreted from an Alteromonas spp. marine strain isolated from the Biscay Bay, targeting the genes involved in its synthesis. First, the genome of this strain was sequenced and different gene clusters related to the synthesis of EPS were identified. Then, a transcriptomic study was carried out to analyse the expression of EPS synthesis related genes in response to glucose and the EPS composition was preliminary characterized.  The long-term objective is to increase our understanding of the patterns of EPS secretion in Alteromonas, which may have a key role in their association with phytoplankton blooms and adaptation to different environmental conditions.

How to cite: Perez-Cruz, C., Estupiñan, M., Llamas-Arriba, M. G., Etxebeste, O., Lanzen, A., and Alonso-Saez, L.: Extracellular polysaccharides (EPS) secreted by a model marine bacterium (Alteromonas spp.), biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-91,, 2020.

Aleksandra Hagberg, Shashank Gupta, Olena Rzhepishevska, Jerker Fick, Mette Burmølle, and Madeleine Ramstedt

Pharmaceuticals and biofilms in a fresh-water stream in the south of Sweden

Aleksandra Hagberg1, Shashank Gupta2, Olena Rzhepishevska1, Jerker Fick1, Mette Burmølle2, Madeleine Ramstedt1

  • 1) Department of Chemistry, Umeå Center for Microbial Research, Umeå University, 901 87 Umeå, Sweden
  • 2) Section of Microbiology, Department of Biology, University of Copenhagen, 2100, Copenhagen, Denmark


Pharmaceuticals have been detected in the aquatic environment all around the globe. The usage of medicine is growing every year, increasing the number of pharmaceutical residues released into the environment. Chronic exposure creates a significant threat to aquatic organisms. For this reason, it is crucial to investigate how pharmaceuticals can affect inhabitants of the aquatic ecosystem. In our study, we aimed to investigate how pharmaceuticals influenced the sessile bacterial species pattern in the Knivsta river in the south of Sweden. By placing the four sampling points before and after contamination (upstream and downstream), we aimed to see differences between locations that were chronically exposed to pharmaceuticals from a local sewage treatment plant and those that remained unexposed. Sampling was made three times in one year. Bacterial populations were analyzed by sequencing 16S RNA. Water chemistry with respect to pharmaceutical content was determined with LC-MS. Bacterial isolates were also collected and showed a range of phenotypes.


How to cite: Hagberg, A., Gupta, S., Rzhepishevska, O., Fick, J., Burmølle, M., and Ramstedt, M.: Pharmaceuticals and biofilms in a fresh-water stream in the south of Sweden, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-84,, 2020.

Torsten Mueller, André Koernig, and Tanja Neumann

The composition, topography, adhesiveness and nanomechanical properties of biomaterials are all factors that affect biological processes, e.g. biofilm formation, cell differentiation, and morphogenesis [1-4]. Atomic Force Microscopy (AFM) is a highly versatile tool, ideal for the characterization of samples properties ranging from single molecules to complex biological systems, on the nm scale.

JPK BioAFMs, like the NanoWizard® ULTRA Speed 2, enable fast imaging of challenging biological samples and the visualization of dynamic processes with high spatio-temporal resolution under near physiological conditions, e.g. the kinetics of collagen type I fibrillogenesis was imaged in situ revealing the formation of the 67 nm D-banding hallmark.

Our distinctive Quantitative Imaging mode (QI™) measures various sample properties such as topography, nanomechanics and adhesion on the nanometer scale. Complex data like contact point, Young´s modulus or recognition images can also be extracted at the same resolution. To demonstrate the capability and flexibility of the QI™ mode, various biological samples like living cells have been investigated and their topographical and mechanical properties determined. QI™, based on fast force mapping, can also be used to determine the mechanical properties of different bacterial strains. We will discuss the application of HighSpeed AFM for the characterisation of dynamic biofilms with high spatiotemporal resolution, information which can then be directly correlated with advanced optical microscopy for immuno-characterisation of the sample.

Investigating large, sticky and rough samples such as tissues and hydrogels using AFM has always been a challenge due to the limited z-axis of the AFM. The HybridStage™, equipped with an extended xyz scanner unit up to 300x300x300 µm3, an additional motorized unit for large sample movements in the mm range and optical tiling, is ideal for investigating such samples. This combination enables multi-region AFM probing over a large, uneven sample area and provides additional correlative optical data sets.

Adhesion dynamics between cells and biomaterials play a crucial role in, e.g. the applicability of potential implant materials. The AFM based Single Cell Force Spectroscopy platform enables quantitative measurement of the interactions between individual cells and any substrate.

A number of important research topics in the field of biomedicine, relate directly to the increased antimicrobial resistance of various biofilms to commonly prescribed drugs, and have identified adhesion, as a leading factor in biofilm formation, colony progression, and pathogenesis of microbial agents. JPK BioAFM has developed novel techniques for studying single-molecule forces and adhesion profiles at the cell/cell or cell/substrate interface. We will provide an overview of how to functionalize various surface substrates for the attachment of bacteria.

We will also provide information on working with AFM under Biosafety Level L2/L3 conditions.


[1]      Elter, P. et al., Eur Biophys J, 2011, 40(3):317-27

[2]      Engler AJ. Et al., Cell; 2006, 126(4):677-89

[3]      Cisneros, DA. et al., Small, 2007, 3(6):956-63

[4]         Koser DA. Et al., Nat. Neurosci.; 2016, 19:1592-1598

How to cite: Mueller, T., Koernig, A., and Neumann, T.: Probing the topography and mechanical properties of biomaterials with atomic force microscopy, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-87,, 2020.

Fabien Lamret, Jennifer Varin-Simon, Sophie Gangloff, and Fany Reffuveille

Bone and joint infections linked to implanted materials are mostly due to Staphylococcus aureus. Deciphering the biofilm structure appears to be a promising strategy to develop antibiofilm molecules in order to curb infection occurrence and the bacterial recurrence. Indeed, the characterization of biofilm architecture and physiology could help to find new therapeutic targets through notable quantification of the matrix main components. Our hypothesis is that the very complex and interconnected bone microenvironment influences the bacterial adhesion and biofilm maturation and so its composition.

To identify the main factors influencing biofilm formation in the bone microenvironment, we determined biofilm biomass and the number of live adhered bacteria in a static model, completed with microscopy approaches to support our results. Different factors of bone microenvironment were tested: starvation, low oxygen rate, excess of magnesium, and presence of bone cell products. Our first results showed that MSSA or MRSA strains did not have the same behaviors under the tested conditions. However, for both types of strains, excess of magnesium combined to paucity of amino acids and oxygen increased the most the proportion of adhered Staphylococcus aureus (a 6 to 43 fold-increase, p = < 0.01). But biofilm biomass quantification and bacterial adhesion results showed divergent profiles leading us to think that matrix could be involved in such contrasts. Scanning electron microscopy highlighted several structures of matrix produced by these bacteria: well-known slime aspect, but also fibrous appearance, and no matrix production was revealed under some conditions. Indeed, all strains produced few matrix when cultured with control medium and oxygenated condition. Only CIP 53.154 strain built a strong slime-like matrix in response to oxygen depletion. However, both MSSA CIP 53.154 and SH1000 strains developed fibrous structures under anaerobic conditions associated with amino acid starvation, high magnesium concentration with or without glucose. MRSA USA300 strain did not seem to produce a matrix under our conditions, which is supported by the literature. Further investigations of the biofilm matrix are needed to conclude on the matrix nature, which surrounds bacteria under our conditions.

The bone microenvironment is complex but our results show that the parameters that mimicked this specific environment influenced the bacterial adhesion and probably the biofilm matrix composition of several strains of Staphylococcus aureus. Further investigations will help to understand how the different factors influence biofilm formation through quantification of the matrix main components by fluorescence microscopy and enzyme digestion. Our final aim is to develop an in vitro model mimicking this specific microenvironment in order to screen different antimicrobial molecules, which could target the biofilm matrix.

How to cite: Lamret, F., Varin-Simon, J., Gangloff, S., and Reffuveille, F.: Staphylococcus aureus biofilm matrix under bone environment influence, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-42,, 2020.

Dishon Hiebner, Caio Barros, Laura Quinn, Stefania Vitale, and Eoin Casey

The contribution of the biofilm extracellular polymeric substance (EPS) matrix to reduced antimicrobial susceptibility in biofilms is widely recognised.  As such, directly targeting the EPS matrix is a promising biofilm control strategy that allows for efficient disruption of the matrix to allow an increase in susceptibility to antibiofilm agents. To this end, engineered nanoparticles (NPs) have received considerable attention. However, the fundamental understanding of the physicochemical interactions occurring between NPs and the EPS matrix has not yet been fully elucidated. An insight into the underlying mechanisms involved when a NP interacts with molecules in the EPS matrix will aid in the design of more efficient systems for biofilm control. The use of highly specific fluorescent probes in confocal laser scanning microscopy (CLSM) to illustrate the spatial distribution of EPS macromolecules within the biofilm is demonstrated. Three-dimensional (3D) colocalization analysis was used to assess the affinity of differently functionalized silica NPs (SiNPs) for specific EPS macromolecules from Pseudomonas fluorescens biofilms. Results show that both the charge and surface functional groups of SiNPs dramatically affect the extent to which SiNPs interact and localize with EPS macromolecules, including proteins, polysaccharides, and DNA. This research not only develops an innovative strategy for biofilm-nanoparticle interaction studies but also provides a platform on which to build more efficient NP systems for biofilm control.

How to cite: Hiebner, D., Barros, C., Quinn, L., Vitale, S., and Casey, E.: Surface Functionalization-Dependent Physicochemical Interactions between Nanoparticles and the Biofilm EPS Matrix, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-93,, 2020.

Monika Astasov-Frauenhoffer, Elena Steiger, Julia Muelli, Olivier Braissant, and Tuomas Waltimo

The aim of the study was to investigate the effect of calcium, magnesium, and zinc on cariogenic biofilm formation and their interaction with bacterial EPS. This was evaluated using two S. mutans strains and different carbohydrates (glucose, sucrose and fructose).

Different combinations of carbohydrates and ions were investigated for their effect on the biofilm formation on hydroxyapatite disks by confocal laser scanning microscopy. Moreover, exopolysaccharides were purified and their affinity to the ions was measured by isothermal titration calorimetry.

The biofilm formation of S. mutans clinical isolate was almost eliminated in the presence of Zn2+ and promoted by Ca2+, while adhesion seems to be more inhibited by Ca2+ and Mg2+ for S. mutans type strain. The EPS of cilincal isolate had a higher binding affinity towards calcium and magnesium than the type strain.

There seems to be a fine balance between these ions that needs to be maintained as excessive concentrations of one or another destroy the balance between the three.

How to cite: Astasov-Frauenhoffer, M., Steiger, E., Muelli, J., Braissant, O., and Waltimo, T.: The role of divalent ions in cariogenic biofilm formation, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-70,, 2020.

The role of extracellular polymeric substances in biofilm community assembly and metabolic functions
Yichao Wu
Bumkyu Kim, Mengfei Li, Patricia Perez-Calleja, and Robert Nerenberg

The membrane-aerated biofilm reactor (MABR) is an emerging wastewater treatment technology that uses O2-supplying membranes as a biofilm support.  Because O2 is supplied from the biofilm base instead of the bulk liquid, MABR biofilms have distinct microbial community structures and behavior.  Past research showed that protozoan predation in MABR biofilms can create a unique void layer at the base of the biofilm. We hypothesized that the void layer could weaken the biofilm and promote sloughing, and investigated this with heterotrophic and nitrifying MABR biofilms.

Biofilms were grown in flat-sheet MABRs (“Base Case”). As a control, a reactor was supplied with cycloheximide in the media to suppress protozoa (“Suppressed Predation”). Each condition was run in triplicate. A rheometer was used to measure biofilm mechanical strength, and MABR flow cells were used to explore detachment. The biofilms were imaged using optical coherence tomography (OCT) (Ganymede, Thorlabs, Germany), and the images were digitally processed to quantify the biofilm thickness and internal void areas. In all tests, the biofilm was first grown to steady-state, as determined by effluent substrate concentrations and biofilm thicknesses.

In the heterotrophic biofilms, predation increased the internal void ratio from 6 ± 7 % to 50 ± 16 %.  The storage modulus was 1,780 ± 1,180 Pa for the Base Case, compared to 9,800 ± 4,290 Pa for Suppressed Predation. Similarly, the loss modulus was 1,580 ± 729 Pa for the Base Case and 363 ± 189 Pa for Suppressed Predation. When subjected to an increased flow, the biofilm loss was 44 ± 24 % for the flow cell with predation, while only 7 ± 9 % for the control.

In the nitrifying biofilms, predation resulted in a greater fraction of internal voids, at 69 ± 6 % for the Base Case vs. 54 ± 5 % for Suppressed Predation. Also, the increased void ratio by predation reduced the biofilm viscosity and elasticity, resulting in greater detachment.

The loss modulus with Base Case and Suppressed Predation was 242 ± 135 Pa and 3640 ± 1860 Pa, respectively. The storage modulus was 1650 ± 853 Pa with Base Case and 23300 ± 11500 Pa with Suppressed Predation. The relative detached area of biofilm with Base Case and Suppressed Predation were 18 ± 12 and 4 ± 5 %, respectively. Thus, the greater detachment for the Base Case was consistent with the weaker mechanical properties.  Predation also decreased the nitrification fluxes and promoted partial nitrification. The selective loss of NOB, as confirmed by fluorescence in-situ hybridization (FISH) and qPCR, may be due to the larger size of AOB clusters, providing greater resistance to predation.

These findings suggest that the effects of protozoa may need to be considered to predict the behavior of heterotrophic and nitrifying MABRs. Also, a better understanding of the microbial ecology of protozoa may lead to more effective MABR operational strategies.

How to cite: Kim, B., Li, M., Perez-Calleja, P., and Nerenberg, R.: Unique effects of predation on heterotrophic and nitrifying membrane-aerated biofilm reactors (MABRs), biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-99,, 2020.