Orals

Across many different habitats, bacteria are often found as sessile communities embedded in a self-secreted matrix of extracellular polymeric substances (EPS)  [1]. The biofilm matrix enhances bacterial resistance to harsh environmental conditions and antimicrobial treatments, and thus hinders our ability to remove detrimental biofilms in medical and industrial applications. Depending on the environmental conditions, biofilms can be found as tethered filaments suspended in flow, known as streamers [2], or surface-attached communities. Despite the importance of the matrix to biofilm survival, little is known about how environmental features shape its microstructure and chemical composition.

Here, we show that a laminar flow of a diluted suspension of Pseudomonas aeruginosa PA14 around a pillar can trigger the formation of suspended filamentous biofilm structures known as streamers and that extracellular DNA (eDNA) plays a fundamental structural role in streamer formation  [3]. We have developed a microfluidic setup that allows real time visualization of the formation of biofilm streamers and the investigation of their biochemical composition by means of lectins staining. Our experiments confirmed that this phenomenon is dominated by the interplay between the viscoelastic nature of EPS, which is extruded by local flow shear, and the secondary flow around the pillar, which promotes the growth of the filaments due to a filtration mechanism. By varying the composition of the biofilm matrix using mutant strains of PA14 and by applying targeted treatment with the enzyme DNase I, we could shed light on the structural role of the different biochemical components: eDNA is essential for streamers formation, while Pel, a positively charged exopolysaccharide which binds to eDNA  [4], affects the filament morphology. In addition, since in this geometry we can study freestanding biofilm filaments  [5], we could probe the shear-induced deformation of streamers to investigate their material properties and reveal that eDNA affects the elastic behaviour of the biofilm matrix, while the viscous behaviour is determine by the quantity of Pel. Finally, thanks to our mechanistic understanding of the interplay between streamers composition and microstructure, we could surprisingly promote streamers formation by adding sublethal concentration of an antibiotic commonly used to treat P. aeruginosa infections. In summary, using the experimental toolbox from biophysics to characterize the biofilm matrix, we could elucidate the relation between chemical composition and microstructure, use our understanding to control streamers formation and gain an insight on this biological system that could make an impact in the medical sector.

[1]      H.-C. Flemming et al., Nat. Rev. Microbiol. 14, 563 (2016).

[2]      R. Rusconi, S. Lecuyer, L. Guglielmini, and H. A. Stone, J. R. Soc. Interface 7, 1293 (2010).

[3]      E. Secchi, G. Savorana, A. Vitale, L. Eberl, R. Rusconi, and R. Stocker, paper in preparation.

[4]      L. K. Jennings et al., Proc. Natl. Acad. Sci. 112, 11353 (2015).

[5]      G. Savorana, R. Rusconi, A. Sartori, L. Heltai, R. Stocker, and E. Secchi, paper in preparation.

How to cite: Secchi, E., Savorana, G., Vitale, A., Eberl, L., Rusconi, R., and Stocker, R.: The role of eDNA in the formation of biofilm streamers, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-10, https://doi.org/10.5194/biofilms9-10, 2020.

Considering the timeline required for the development of novel antimicrobial drugs, increased attention should be given to repurposing existing drugs and improving their antimicrobial efficacy, particularly for chronic infections associated with biofilms. Methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) are common causes of biofilm-associated infections however each species has a distinct biofilm phenotype resulting in different biofilm matrix characteristics.. Nanoparticles (NPs) have the potential to significantly enhance the delivery of antimicrobial agents into biofilms, however the physicochemical properties which influence these interactions between NPs and the biofilm are not fully understood. The influence of NP surface chemistry on interactions with MRSA and MSSA biofilms was explored in this study. Mesoporous silica nanoparticles (MSNs) with different surface functionalizations (bare-B, amine-D, carboxyl-C, aromatic-A) were synthesised. Following interaction studies, MSNs were loaded with vancomycin (VAN) to observe biofilm eradication. The two negatively charged MSNs (MSN-B and MSN-C) showed a higher VAN loading in comparison to the positively charged MSNs (MSN-D and MSN-A). Cellular binding with MSN suspensions (0.25 mg mL^-1) correlated with reduced viability of both MSSA and MRSA biofilm cells. MSNs were shown to be efficient carriers of vancomycin while also displaying significantly improved efficiency compared to free VAN. This allowed the administration of low MSNs concentrations, while maintaining a high local concentration of the antibiotic surrounding the bacterial cells, indicating a promising novel therapeutic approach for S. aureus biofilm infections.

How to cite: Devlin, H., Fulaz, S., Vitale, S., Quinn, L., O'Gara, J., and Casey, E.: Tailoring nanoparticle-biofilm interactions to increase efficacy of antimicrobial agents against Staphylococcus aureus , biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-11, https://doi.org/10.5194/biofilms9-11, 2020.

Effective diffusion coefficients are often required for kinetic descriptions of biofilms. Many previous studies have measured diffusion coefficients for specific molecule-biofilm combinations. As a result, many biofilm researchers today rely on literature values of diffusion coefficients for their own biofilm system. However, the reported diffusion coefficients in literature fall within a wide range, even for the same molecule. One potential cause of this range is the accuracy of the methods used to measure diffusion coefficients. The objective of this study was to determine the precision (similarity between repeated experiments) and bias (difference between measured and true diffusion coefficient) of six common methods. The six selected methods were based on determining mass balances and on microelectrode measurements. The precision and bias were quantified based on mathematical models of the six methods, with oxygen diffusion in granular sludge as a case study. The precision was assessed by a Monte Carlo uncertainty analysis, which considers the propagation of uncertainty in the input experimental parameters. The bias was determined for six potential sources of error: solute sorption, biomass deactivation, a concentration boundary layer, granule roughness, granule shape, and granule size distribution. From the Monte Carlo analysis, it followed that the precision of the methods ranged from 4-77% relative standard deviation. The microelectrode methods were more accurate than the mass balance methods. The bias due to the combined effect of the six errors was an underestimation of the diffusion coefficient by 74%. This shows that current methods are unable to accurately determine diffusion coefficients. We do not propose improvements to the current methods, but instead discuss why inaccurate diffusion coefficients are sufficient for accurate engineering of biofilm processes. 

How to cite: van den Berg, L., van Loosdrecht, M., and de Kreuk, M.: How to Measure Diffusion Coefficients in Biofilms: A Critical Analysis, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-85, https://doi.org/10.5194/biofilms9-85, 2020.

Rhizobium leguminosarum synthesizes an acidic polysaccharide formed by the polymerization of octasaccharide repeating units containing glucose (Glc), glucuronic acid (GlcA) and galactose (Gal) in a 5:2:1 ratio with particular substitutions; most of it is secreted to the extracellular medium (EPS) and part of it is retained on the bacterial surface as a capsular polysaccharide (CPS). Rap proteins, substrates of the PrsDE type I secretion system (TISS) share at least one Ra/CDHL (cadherin-like) domain and are involved in biofilm and matrix development either by cleaving the polysaccharide (Ply glycanases) or by altering the bacterial adhesive properties. Previous studies have shown that RapA2 is a monomeric calcium-binding lectin capable of binding specifically the R. leguminosarum CPS through a Ra/CDHL domain. It was shown that the absence or excess of RapA2 in the extracellular medium alters the biofilm matrix’s properties.

In this work we identified a new Rap protein (RapD), which comprises an N-terminal Ra/CDHL domain and a C-terminal domain of unknown function. By Western blot analysis using specific polyclonal antibodies we showed that in planktonic cultures RapD is co-secreted with the other Rap proteins in a PrsDE-dependent manner. Furthermore, under conditions that favor EPS production, a prominent RapD secretion was observed. In addition, colony blot assays indicated that RapD is associated with the biofilm matrix.  Interestingly, size exclusion chromatography of the EPS produced by the ΔrapA2 ΔrapD double mutant showed differences in the EPS profiles compared with those of the single mutants and the wild type strain, thus suggesting a functional interaction between the RapA2 and RapD proteins.

 Biophysical studies showed that calcium triggers proper folding and multimerization of recombinant RapD. Besides, further RapD conformation changes were observed in the presence of EPS.

ELISA and BIA (binding inhibition assay) assays showed that in the presence of calcium, RapD specifically binds the EPS and that galactose residues would be involved in this interaction.

In conclusion, RapD is a multimeric calcium-dependent EPS lectin that is co-secreted with the other Rap proteins via TISS PrsDE. Unlike RapA2, RapD is not retained on the bacterial surface but would rather interact with the released EPS. Finally, our results suggest that the interaction of RapA2 and RapD with the CPS or the EPS somehow affects the polysaccharide processing and therefore the biofilm matrix.

How to cite: Tarsitano, J., Russo, D. M., Alonso, L., and Zorreguieta, A.: A multimeric matrix-associated lectin (RapD) affects proper exopolysaccharide processing in Rhizobium leguminosarum , biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-152, https://doi.org/10.5194/biofilms9-152, 2020.

Biofilms are highly structured, densely packed bacterial consortia where many different species can coexist. During biofilm development and growth, the different species often form spatial distribution patterns that govern biofilm composition and function. In some cases, emerging structures have been explained as the result of social interactions between bacteria, e.g. cooperation and competition. Others emphasize the role of local mechanics, where spatial structuring arises from forces exerted between cells or between cells and their environment. Typically, these two lines of argumentation are treated separately. Here, we show that mechanics and social interactions can be strongly interrelated and their combination can crucially impact biofilm formation and dynamics. Using confocal microscopy and bacterial co-culture assays, we examine how bacterial antagonism impacts biofilm mechanics, and vice versa. We study competing Vibrio cholerae strains that kill on contact using the Type 6 secretion system. In case of mutual killers, i.e. two V. cholerae strains that can kill each other on contact, this social interaction leads to the formation of clonal domains of the competing strains (Mc Nally et al., Nat Commun, 2017). Intuitively, an unequal fight may enable a superior killer to invade and quickly eliminate a much weaker competitor. However, we observe that killer cells can coexist with killing-deficient target cells for very long times, and find that this results from the mechanical consequences of the deadly competition. Killing produces dead cells, which accumulates between domains of competing cells and prevents subsequent killing. Counterintuitively, our results suggest that antagonistic interactions stabilize coexistence in diverse communities. The findings demonstrate that the impact of social interactions in bacterial consortia is complex, requiring the understanding of the structural and the statistical-mechanical processes in biofilms.

How to cite: Steinbach, G., Crisan, C., Ng, S. L., Hammer, B., and Yunker, P.: Interplay of microbial interaction and biofilm mechanics govern biofilm dynamics, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-112, https://doi.org/10.5194/biofilms9-112, 2020.