Posters

While in biofilms bacteria are embedded into an extracellular matrix which forms inaccessible barrier for antimicrobials thereby drastically increasing the concentrations of antibiotics required for treatment. Here we show that the susceptibility of S. aureus and P. aeruginosa to antibiotics in mixed biofilms significantly differs from monoculture biofilms depending on both conditions and chosen antimicrobial agents. While S. aureus could completely avoid vancomycin, ampicillin and ceftriaxone by embedding into the biofilm of P. aeruginosa, the very same consortium was characterized by 10–fold increase in susceptibility to broad-spectrum antimicrobials like ciprofloxacin and aminoglycosides compared to monocultures. These data clearly indicate that efficient treatment of biofilm-associated mixed infections requires antimicrobials active against both pathogens, since the interbacterial antagonism would enhance the efficacy of treatment. Moreover, similar increase in antibiotics efficacy was observed when P. aeruginosa suspension was added to the mature S. aureus biofilm, compared to S. aureus monoculture, and vice versa. These findings open promising perspectives to increase the antimicrobial treatment efficacy of the wounds infected with nosocomial pathogens by the transplantation of the skin residential microflora.

This work was supported by the Russian Science Foundation (Project №20-64-47014)

How to cite: Trizna, E., Baidamshina, D., Yarullina, M., Mironova, A., Khabibrakhmanova, A., Kurbangalieva, A., and Kayumov, A.: Bidirectional alterations in antibiotics susceptibility in Staphylococcus aureus - Pseudomonas aeruginosa dual-species biofilm, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-100, https://doi.org/10.5194/biofilms9-100, 2020.

Mechanisms of electron transfer vary greatly within the diverse group of electroactive microorganisms and so does the need to attach to the electrode surface, e.g. by forming a biofilm.

Electrochemical impedance spectroscopy (EIS) and confocal laser scanning microscopy (CLSM) are well established methods to monitor cell attachment to an electrode surface and have therefore been combined in a flow cell as a screening system. The flow cell, equipped with a transparent indium tin oxide working electrode (ITO WE), allows monitoring of attachment processes in real time with minimal needs for additional biofilm preparation. In preliminary experiments the flow cell was successfully used as microbial fuel cell (MFC) with a potential of +0.4 V vs. Ag/AgCl using Shewanella oneidensis as electroactive model organism. [1]

Commonly, graphite-based electrode materials are used in bioelectrochemical systems due to their low costs and high conductivity. However, the hydrophobic and negatively charged surface is not yet optimal for microbial attachment. There are numerous attempts on electrode surface engineering in order to overcome this problem. In the majority of studies the biofilm analysis and evaluation of the attachment takes place at the end of the experiment, neglecting the impacts of the chemical surface properties and initial electrode conditioning during the very beginning of biofilm formation.

To investigate initial attachment and biofilm formation in real-time, the transparent ITO-electrode is coated with polyelectrolytes differing in hydrophobicity and polarity to evaluate their effects on the initial surface colonisation by different electroactive microorganisms. Combining CLSM and EIS, both, surface coverage and electrochemical interaction of electrode-associated bacteria can be assessed.

With this we aim to understand and ease initial steps of biofilm formation to improve efficiency of bioelectrochemical applications, e.g. with regards to start-up time.

[1] Stöckl, M., Schlegel, C., Sydow, A., Holtmann, D., Ulber, R., & Mangold, K. M. (2016). Membrane separated flow cell for parallelized electrochemical impedance spectroscopy and confocal laser scanning microscopy to characterize electro-active microorganisms. Electrochimica Acta, 220, 444-452.

How to cite: Frühauf, H., Stöckl, M., and Holtmann, D.: Finding the comfort zone: Online-monitoring of electroactive bacteria colonising electrode surfaces with different chemical properties, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-46, https://doi.org/10.5194/biofilms9-46, 2020.

Shewanella oneidensis MR1 is the best understood model organism with regards to dissimilatory metal reduction and extracellular electron transfer onto carbon electrodes in bioelectrochemical systems (BES)¹. However, under anoxic conditions S. oneidensis is known to form very thin biofilms resulting in low current density output. In contrast, another exoelectrogenic model organism Geobacter surfurreduscens can form electroactive biofilms up to 100 µm in thickness. This organism is known for its ability to transport electrons over a long range (> 10 µm) along a network of protein filaments, called microbial nanowires. Although still controversial, it was recently reported that OmcS has a special importance for the conductivity of these nanowires². One of the key differences between G. surfurreduscens and S. oneidensis lies in how cell-to-cell electronic communication occurs, which dictate the range of electronic communication between distant cells. S. oneidensis relies on direct cell-to-cell communication via electron transfer between outer membrane cytochromes or via soluble redox active flavins that are secreted by the cells³. Our research is based on the question, what if the S. oneidensis biofilm formation could be improved by introducing an artificial electronic network, similar to the native microbial nanowires for G. sulfurreducens?

We hypothesize that synthetic biofilms containing conductive nanostructure additives would allow S. oneidensis to build multilayer thick biofilms under anoxic conditions on solid electron acceptors. To answer this question of how conductive materials affect the formation of anoxic S. oneidensis biofilms, we integrated both biological and synthetic conductive nanostructures into these biofilms. As biological additive, the c-type cytochrome OmcS purified from G. sulfurreducens was utilized. As synthetic additives, both commercially available biotinylated gold nanorods and in-house electrochemically synthesized metal nanostructures were added to anoxic S. oneidensis biofilms.

Cultivation and characterization of the biofilms was performed using our newly developed microfluidic bioelectrochemical platform. Microbial cultivation with the aid of microfluidic flow chambers has a great potential to form biofilms on an easy to handle laboratory scale with simultaneously ongoing multianalytical analysis⁴. In our bioelectrochemical microfluidic, system S. oneidensis biofilms can be grown under anoxic conditions using an anode as sole electron acceptor. The growth behavior and bioelectrochemical performance was evaluated by a combination of electrochemical techniques (chronoamperometry, electrochemical impedance spectroscopy, cyclic voltammetry) and optical analyses (confocal laser scanning microscopy and optical coherence tomography). The strategy of conductive nanostructured additives for improved electroactive biofilm formation could be an important tool for other exoelectrogenic microorganisms in order to exploit their physiological abilities for biotechnology.

References:

How to cite: Klein, E., Wurst, R., Rehnlund, D., and Gescher, J.: Interfacing anoxic Shewanella oneidensis biofilms with electrically conducting nanostructures, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-139, https://doi.org/10.5194/biofilms9-139, 2020.

Introduction: Biofilms are 3-dimensional (3D) aggregates of microorganisms that are associated with a wide range of diseases. Although there have been several studies investigating biofilm formation on two-dimensional substrates, the use of 3D substrates may result in more representative and clinically relevant models. Accordingly, the aim of this study was to compare the growth of biofilms in the 3D substrates against biofilms grown in 2D substrates.
Material and Methods: Two grams of medical grade polycaprolactone (PCL) were loaded into a plastic Luer-lock 3 ml syringe and a 23G needle was used as a spinneret. The syringe was placed in a melt electro-writing (MEW) device to obtain fine fibers under controlled parameters. The 3-dimensional MEW PCL scaffolds were manufactured and characterised with an overall thickness of ~ 0.8 mm, with ~ 15 μm diameter fibers and ordered pore sizes of either 100 or 250 µm. PCL films employed as 2D substrates were manufactured by dissolving 10 gms of PCL in 100 ml chloroform and stirred for 3 h to obtain a transparent solution. Then, the solution was cast in glass petri dishes and dried to remove all organic solvents. In addition, commercial hydroxyapatite discs were also used as 2D controls. Unstimulated saliva from six healthy donors (gingival health) were used to grow biofilms. The formed biofilms were assessed at day 4, day 7 and day 10 using crystal violet assay, confocal microscopy, scanning electron microscopy and next-generation 16s sequencing.
Results: The results demonstrates that 3D PCL scaffolds dramatically enhanced biofilm biomass and thickness growth compared to that of the 2D controls. Confocal microscopy of biofilms at day 4 stained with SYTO 9 and propidium iodide showed thickness of biofilms in 2D substrates were 39 µm and 81µm for hydroxyapatite discs and PCL films, respectively. Biofilms in 3D substrates were 250 µm and 338 µm for MEW PCL 100µm pore size and MEW PCL 250 µm pore size, respectively. Similar results were noticed at day 7 and day 10. Scanning electron microscopy showed biofilm bridges formed over the fibers of the MEW scaffolds. Pilot trials of next generation sequencing detected similar taxa in biofilms formed in 3D scaffolds compared to that of 2D substrates.
Discussion: We have successfully investigated a 3D biofilm growth model using 3D medical grade PCL scaffolds. Thicker biofilms can be conveniently grown using this inexpensive static model. This will facilitate 3D microbial community studies that are more clinically relevant and improve our understanding of biofilm-associated disease processes.

How to cite: Ramachandra, S., Abdal-hay, A., Han, P., Lee, R., and Ivanovski, S.: Melt electro written three-dimensional scaffolds engineered as oral microcosm models-an in vitro study., biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-29, https://doi.org/10.5194/biofilms9-29, 2020.

Chronic wounds, for instance venous, pressure, arterial and diabetic ulcers, are a major health problem throughout the world. Compared with normal wounds, those that take more than four weeks to heal are defined as chronic. Interestingly, the numbers of patients suffering from chronic wounds and the cost for treatment have been increasing during the past two decades. There is increasing evidence that suggests that bacteria infect those chronic wounds and there exist as a biofilm, which affects wound healing and success of treatment. To study biofilms in infected wounds, both in vitro and in vivo biofilm models are important to be developed.

In this project, a dynamic ex vivo chronic wound biofilm model for Staphylococcus aureus using a 3D printed chamber and porcine skin was developed. This dynamic model then used to determine antibiotic treatment by using poly(ε‐caprolactone) (PCL) electrospun fibrous mats containing different antibiotics, e.g. tetracycline, gentamicin and fusidic acid. Furthermore, electrospun PCL/silk fibroin scaffolds were also used as carrier of gentamicin. The killing effect of mature S. aureus MRSA 252 growing in the wound model was tested by both viable count and qPCR.

The results indicated that this newly designed dynamic model was successful in mimicking single-strain biofilm on infected chronic wounds. Compared with traditional biofilm assays, the flow system generates an air-liquid-solid interface, which more closely approaches to real conditions. Furthermore, results from using electrospun fibrous scaffolds provided strong evidence for their potential in clinical applications to treat infected skin.

How to cite: Cheng, Y., De Bank, P., and Bolhuis, A.: Modelling Staphylococcus aureus biofilms on infected chronic wounds, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-82, https://doi.org/10.5194/biofilms9-82, 2020.

The physical structure and mechanical properties of a biofilm are known to respond to external stressors, such as hydrodynamic shear, and are expected to play a vital role in determining biofilm-associated drag. Yet, both, how these structural and mechanical properties interact with one another and with fluid flow, and how these interactions influence drag is poorly understood. In part this is due to a lack of standard methods for studying biofilm physical and mechanical (physico-mechanical) properties and relating them to drag. To date, rigid structures, such as sandpaper, have been typically used to model biofilms. Whilst rigid structures can simulate roughness, they neglect features such as viscoelasticity and heterogeneity. To address this, our novel work demonstrates the practical application of new test methods for biofilm research: the use of elastomeric and gel-like materials to better model biofilm physico-mechanics under controlled flow conditions, and the use of tensile and rheological testing to measure the elastic modulus of marine biofilms. Artificial biofilms were cast / made from materials with mechanical properties comparable to natural biofilms. Marine biofilms were grown in-house, within a recirculating system, using a field-sourced, mixed species inoculate. The elastic modulus of marine and artificial biofilms was measured using tensile and rheological testing.  Though elastic modulus has been recorded for biofilms previously, until now, the elastic modulus of marine biofilms had not been recorded; partly due to the complexity of their physical structures and their biological composition (bacterial and microalgal components). Despite biological differences, the elastic modulus of marine biofilms tested sits comfortably within the range recorded for other biofilms studied, at 0.0000098 MPa - 0.0002 MPa. A marine biofilm flow cell was utilised for pressure drop experiments, alongside the use of a non-invasive imaging technique, Optical Coherence Tomography. This experimental set-up enabled real-time visualisation and data collection of the physical response of the elastomers and biofilms grown in the marine environment to different flow rates. We found that for artificial biofilms, elasticity had a greater impact on biofilm-associated drag than roughness (P < 0.05). Biofilms are a unique and complex material, and therefore to better understand their physico-mechanical properties in flow, we first need to understand these properties independent of their complex biology. The use of fully artificial biofilms, with controlled properties, based on mechanical properties of marine biofilms, can help achieve this.

How to cite: Jackson, A., An, S., Dennington, S., Stoodley, P., Wharton, J., Longyear, J., and Webb, J.: Using elastomeric materials to model biofilm physico-mechanical properties and the associated drag penalty, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-61, https://doi.org/10.5194/biofilms9-61, 2020.