Posters

Diabetes increases the blood glucose levels above those of healthy individuals and poorly controlled diabetes is associated to ketoacidosis. Different authors have shown evidences that diabetes is linked to a higher risk of developing infections in different parts of the body. Although the reasons why diabetes enhances infection episodes are not entirely clear, different undesired physiological responses under diabetic environments are pointed out as the main causes, for example, inflammatory reactions, poor vascularization, neutrophilic chemotaxis or phagocytosis. However, it has so far not been quantified how high concentrations of glucose and ketone bodies can affect the beginning of the infectious process, i.e. the formation of biofilms.

In this sense, this research will address how the presence of glucose and ketone bodies can alter the biofilm formation capacity of Staphylococcus aureus. The research will be carried out with six different diabetic conditions, including the individual action of both components (glucose and ketone bodies) and the combined action.

The main conclusion of this work is that any studied diabetic condition is able to increase the slime index of S. aureus with respect to control (bacteria grown without diabetic supplements), so the biofilm formation capacity of this bacterium would rise in diabetic people. In addition to the change that can be as high as 400% in glucose concentrations of 1.9 mg/ml, the clustering behavior among the bacteria is also modified at all condition differently.

How to cite: Fernández Grajera, M., Fernández Calderón, M. C., Pacha Olivenza, M. A., Pérez Giraldo, C., Gallardo Moreno, A. M., and González Martín, M. L.: Biofilm formation capacity of S. aureus under diabetic environments, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-141, https://doi.org/10.5194/biofilms9-141, 2020.

Background: The use of different chemicals for agriculture, industry and mining has caused pollution of agrarian soils which provokes changes in rhizosphere microflora. We have studied 10 bacterial strains isolated from a winter wheat Cd-polluted field in Ukraine by their taxonomic position, biochemical properties and resistance to 3 classes of toxicants: heavy metals (Cu²⁺, Cd²⁺), non-metals (perchlorate-ion), organic xenobiotic (1-chloro-4-nitrobenzene, CNB).  

Objectives: To study the effect of toxicants on the biofilm–formation ability of individual strains and a mixed community.

Methods: Biofilm characteristics (total microcosm growth, biofilm strength and attachment to the microcosm walls) were studied by combined biofilm assay (n = 4) with four treatments including 100 mg/L Cu²⁺, 25 mg/L Cd²⁺, 300 mg/L ClO₄^-, and 100 mg/L CNB, with correlations and Principal component analysis (PCA) used to investigate data.

Results: We found that microbial community had a greater resistance to the toxicants compare to individual strains. The presence of heavy metals increased the strength of biofilms, and in most cases growth and biofilm strength were positively correlated. However, in the presence of Cd²⁺ this correlation was lost. Perchlorate affected bacteria, increasing mucus production and biofilm strength though it also reduced attachment levels. Finally, we also found that CNB could be used as a source of Carbon and energy during biofilm–formation.

How to cite: Iungin, O. and Spiers, A.: Chemical stress contribution in bacterial biofilm formation, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-49, https://doi.org/10.5194/biofilms9-49, 2020.

Understanding the interplay between hydrodynamics and biogeochemical processes is of growing importance in environmental applications and studies, especially in the fields of bioremediation and ecology. The majority of the microbial communities living in soil have a surface-attached lifestyle, allowing them to form biofilms. The biofilm growth influences pore geometries by clogging them and thus redirecting the flow, which in return affects biofilm development and local mass transport. After initially clogging single pores, the biofilm structure expands to larger clusters before eventually clogging the porous medium entirely. We study these processes with a soil-born microorganism, Bacillus subtilis, in microfluidic devices mimicking porous media to get a mechanistic understanding of the driving factors of bioclogging of porous media on different scales.

Carefully designed porous geometries were used for the experiments to study biofilm growth under different flow conditions. After being seeded with bacteria, devices were exposed to a continuous nutrient flow during several days. Continuously monitoring the pressure evaluation and imaging the biofilm growth using Brightfield microscopy allowed a high temporal resolution of biofilm growth processes.

An interplay of hydraulic parameters and geometric features of the porous medium as well as the mass flow rate of nutrients drive the speed of pore clogging. Besides the pore scale clogging, the initiation of biofilm formation as well as the speed of clogging of the entire medium are influenced by the before mentioned parameters. Furthermore, the size and number of the biofilm clusters formed seem to drive the medium scale clogging. This leads to inverting trends concerning the clogging rate in one pore when compared to the porous medium scale for different pore sizes. These results shed light on the pore-scale mechanism as well as driving parameters of biofilm formation and bioclogging and their transferability to the next larger scale, e.g. a porous medium.

How to cite: Kurz, D., Secchi, E., Stocker, R., and Jimenez-Martinez, J.: Driving factors for bioclogging of pores and porous media, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-23, https://doi.org/10.5194/biofilms9-23, 2020.

In this contribution we present results on non-destructive chemical imaging in the mid-infrared (MIR) region of well-defined biofilms formed by Pseudomonas simiae. Biofilms were grown on stainless steel slides using a static biofilm model (incubation lasted for seven days at 10 °C, with repetitive medium changes). The MIR spectra correlate with fundamental molecular vibrations and are therefore characteristic for chemical composition and structure [1, 2]. Besides a brief insight into the systematic of how the investigated biofilms were grown the main focus will be on MIR spectroscopic measurements including dynamic observation of drying processes of bacteria, as well as spatially resolved scans of the steel plates with an MIR microscope. The obtained hyperspectral chemical images of biofilms were analyzed by various spectroscopic data analysis techniques.

Furthermore, the dynamic spectroscopic observation of the drying process of planktonic Pseudomonas simiae cultures in nutrient solution gave insight in dynamic variances in certain functional chemical groups of the bacteria.  These variances have also been observed in biofilm samples and may correlate with vitality. The MIR chemical images, where each pixel is composed of an entire MIR spectrum (4000-400 cm^-1) provide detailed information of the investigated biofilms such as their composition and spatial structure. The overlay with conventional microscope images relates spectroscopic to visual data, both laterally resolved in the µm-range, over a scan area of up to 10 x 40 mm².

The variation of the vibrational bands was screened, revealing high and low variance bands, to identify certain spectral regions suitable for classification of the investigated biofilm samples. Characteristic spectral bands were found and related to data from literature. Furthermore, differences in the spatial distribution of proteins and carbohydrates as part of the bacteria and extracellular polymeric substances were clearly identified.

Acknowledgment: 
This work was created within a research project of the des Austrian Competence Centre for Feed and Food Quality, Safety and Innovation (FFoQSI). The COMET-K1 competence centre FFoQSI is funded by the Austrian ministries BMVIT, BMDW and the Austrian provinces Niederoesterreich, Upper Austria and Vienna within the scope of COMET -Competence Centers for Excellent Technologies. The programme COMET is handled by the Austrian Research Promotion Agency FFG.

References:
[1] Andreas Schwaighofer, Markus Brandstetter and  Bernhard Lendl , “Quantum cascade lasers (QCLs) in biomedical spectroscopy”, Chem. Soc. Rev. 46, 5903-5924 (2017)
[2] Jakob Kilgus, Gregor Langer, Kristina Duswald, Robert Zimmerleiter, Ivan Zorin, Thomas Berer, and Markus Brandstetter, "Diffraction limited mid-infrared reflectance microspectroscopy with a supercontinuum laser," Opt. Express 26, 30644-30654 (2018)

How to cite: Leiss-Holzinger, E., Zimmerleiter, R., Wagner, E. M., Rychli, K., and Brandstetter, M.: Dynamic and spatially resolved mid-infrared characterization of biofilms, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-92, https://doi.org/10.5194/biofilms9-92, 2020.

Filtration through natural biofilms in Rapid Sand Filters (RSFs) is among the most used processes to remove ammonium and manganese from groundwaters^[a]. However, initial biofilm seeding is relatively slow^[b], and little is known about the spatial-temporal distribution of the activities. The objectives of this work were to: (a) understand heterogeneity of microbial populations and activities in depth and time, (b) discover how it impacts the process, and (c) develop a mathematical model to propose and experiment enhanced “start-up” strategies.

A stainless-steel column filled with sand was fed with groundwater, with the possibility to modulate inlet temperature and substrate concentrations in “enhanced configuration”. Ammonium, nitrite and manganese concentrations were measured by spectrophotometry at the inlet, at several intermediate column heights and outlet of the RSF. A model was developed with Aquasim^[c] software where biological reactions, and evolutions of soluble compounds, free and attached functional microbial populations are described. Sand and water at different experiment stages and in RSF depths were sampled for DNA extraction and 16S rDNA sequencing, qPCR and metagenomic analysis.

Results shed light on heterogeneity in time of the activities: nitrification systematically begins before manganese oxidation. Analysis in all depth of the RSF, “profiles”, show that all activities are evenly distributed during the seeding and attachment of planktonic microorganisms. However, after the initial phase “start-up”, profiles indicate logically that the biological activities migrate to the inlet of the RSF where substrates are. Most of the substrates are oxidized on the first quarter of sand media depth.

Relative abundances of microorganisms indicate that active species changed from the start-up phase to the production phase: AOB Nitrosomonas species were dominant during ammonium oxidation, while commamox Nitrospira species were mostly found in production.

The model fits pilot data in terms of elimination periods duration and distribution of the activities and allowed to estimate parameters to further simulate “start-up” configurations. By increasing temperature and substrates loading rate, effective nitrifying biofilm settlement was achieved 4.7 times faster than in conventional conditions. However, no significant improvement was observed for manganese oxidation.

At the end of the start-up phase, with both conventional and accelerated method, the filters hosted similar communities. The model, confronted to experiments in production time, validated that the spatial heterogeneity in depth of the RSF ensures robustness of the biological process to punctual over charge of ammonium and manganese.

Our study showed that (a) spatial-temporal heterogeneity is linked to growth of different microbial populations in time, but also related to local conditions in time and depth, and (b) heterogeneity in depth is a characteristic of RSFs and is responsible for robustness and resilience of the process.

REFERENCES

[a] Tekerlekopoulou et al. J Chem Technol Biotechnol 88 (5), 751–773, 2013.

[b] Cai, et al. Bioresource Technol., 176, 149–155, 2015.

[c] Reichert. Environmental Software 10 (3), 199-210, 1995. 

How to cite: Etcheberry, T., Peyre Lavigne, M., Trias, R., Paul, E., and di Gioia, L.: Dynamics of biofilm spatial-temporal heterogeneity in RSFs for ammonium and manganese removal from groundwaters, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-103, https://doi.org/10.5194/biofilms9-103, 2020.

Stenotrophomonas maltophilia is one of the most frequently isolated multidrug resistant opportunistic pathogens. It contributes to disease progression in cystic fibrosis patients and is found in wounds, other infected tissues and on catheter surfaces. Only little is known on key processes linked to biofilm formation of S. maltophilia on a broader basis. Thus the aim of this study was the identification of key processes involved in biofilm formation of S. maltophilia on a global level. Therefore, we analyzed biofilm profiles of 300 globally collected clinical and environmental isolates of the main and recently identified lineages Sgn3, Sgn4 and Sm2 - Sm18 (Groeschel et al., 2020). These data together with the 3D structural analysis of a subset of clinical 40 clinical isolates revealed an unexpectedly high phenotypic variability on a strain specific level. Further transcriptome analysis of seven clinical isolates using biofilm grown cells identified a set of 106 shared and coexpressed genes and roughly 30-35 strain-specific genes. Based on these findings S. maltophilia employs a mostly fermentative growth modus under the biofilm conditions and uptake of iron, phosphorous and other metals appears to be of high relevance. Surprisingly, the transcriptome profiles of biofilm versus planktonic cells revealed that only 8.6% of all genes were differentially regulated when both conditions were compared.  This implies that only relatively few genes contribute to the change from planktonic to biofilm life style. Thereby iron uptake appears to be a key factor involved in this metabolic shift. The transcriptome data generated in this study together with the phenotypic and metabolic analysis represent so far the largest data set on S. maltophilia biofilm versus planktonic grown cells. This study now lays the foundation for the identification of new strategies in fighting S. maltophilia infections in clinical settings.

Ref:  Gröschel et al., 2020 ,The phylogenetic landscape and nosocomial spread of the multidrug-resistant opportunist Stenotrophomonas maltophilia. Nature Commun. 2020 Apr 27;11(1):2044. doi: 10.1038/s41467-020-15123-0.

How to cite: Alio, I., Gudzuhn, M., Schölmerich, M., Pérez García, P., Vollstedt, C., Mamat, U., Poehlein, A., Steinmann, J., Kohl, T., and Streit, W.: Global analyses imply that Stenotrophomonas maltophilia biofilms are phenotypically highly diverse despite a common transcriptome profile, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-73, https://doi.org/10.5194/biofilms9-73, 2020.

Pancreatic cancer is the fourth leading cause of cancer death worldwide. The most common sign of presentation of pancreatic cancer is obstructive jaundice, which prevents the drainage of bile into the intestines and it is often associated with decreased survival in patients. Nowadays more than 70% of the patients with biliary obstructive jaundice is treated by biliary stenting; however, biliary stenting disrupts the natural anatomic barrier between the biliary and the gastrointestinal tract, strongly increasing the risk of a bacterial infection. Moreover, duodenal bacteria, by gaining access into the biliary system, can adhere to the stent surface and develop biofilms. Nevertheless, very little is known about the growth of biofilms on the stents and their role in infectious post-operative complications. In particular, the biliary system is an inherently fluid mechanical environment, where the gallbladder provides the driving pressure and the flow rate of the bile going through the ducts depends on the resistance between the gallbladder and the downstream end of the common bile duct. The average flow rate of the bile ranges between approximately 0.5 to 5 ml/min, which depends if the body is fasting or after a meal; this flow rate then corresponds – in the case for example of plastic stents, which are typically 2-4 mm in luminal diameter – to a maximum flow velocity of about 1-40 mm/s and to a shear rate at the inner surface of the stent of 1-80 s^-1. Therefore, the mechanical stress induced by the bile flow in the stent is likely to play a significant role in the formation of biofilms, as shown by our data. Six clinically relevant isolates from preoperative biliary stents were selected to be grown inside microfluidic channels at different flow rates, in which bacterial attachment and biofilm dynamics were recorded and quantified. We found that fluid flow largely influences biofilm morphology in all the isolates, for which the conditions of flow and shear stress that trigger heterogeneities in biofilm structure have been determined. These results will help us to improve our understanding of biofilm formation in the presence of fluid dynamic environments and eventually consider optimal parameters of flow in the design of medical devices.

How to cite: Cardenas, C. and Rusconi, R.: Heterogeneities in biofilms from clinical isolates under flow conditions, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-113, https://doi.org/10.5194/biofilms9-113, 2020.

Biofilms are bacterial communities embedded in an extracellular matrix, able to adhere to surfaces. A deeper knowledge of the biofilm as a whole will aid the development of efficient methods to control deleterious biofilms (clinical biofilms, biofouling) or to enhance beneficial ones (waste-water treatment, bio-filtration). P. fluorescens has been widely studied, this strain produces bioactive secondary metabolites, and forms biofilms[1]. Several experimental set-ups have been widely used for in vitro biofilm cultivation of P. fluorescens, even if a deep characterization among different culture conditions is still lacking in the literature. This work, based on previous studies[2], is focused on the investigation of growth conditions on biofilm structure and properties. Growth kinetics of P. fluorescens biofilms was characterized in vitro under stagnant and flow-controlled conditions, using a rotating annular bioreactor. Two different supports in borosilicate glass and polycarbonate have been used. Bacterial growth kinetics has been measured through bio-turbidity analysis and TOC/DOC quantification. Biofilm morphology has been quantified through optical microscopy and image analysis by measuring the fraction of support surface covered by biofilm. The wetting properties of the biofilm layers have been investigated by using an innovative device, named Kerberos®, able to control centrifugal and gravitational forces acting on a single droplet placed on a surface[3]. The evolution of the droplet shape and position was measured as function of the imposed stress, to quantify wetting of different biofilm coated samples, following already assessed methodologies[4]. Different chemo-physical environments, investigated by changing growth medium, physical support, and imposed flow stress, induced different growth kinetics, biofilm morphology, and wetting properties. Accurate experimental measurements allowed us to estimate in a quantitative way the influence of investigated parameters on specific morphologic measurements.

How to cite: Castigliano, M., Petala, M., Kostoglou, M., Guido, S., Caserta, S., and Karapantsios, T.: Pseudomonas Fluorescens biofilm in rotating annular bioreactor: formation kinetics and wetting properties, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-145, https://doi.org/10.5194/biofilms9-145, 2020.

Biofilms consist of bacteria immobilized in extracellular polymeric substances (EPS) with a complex three-dimensional morphology. This inevitably results in gradients (concentration, cell count, pH, etc.) directly affecting the overall behavior of biofilms ¹. Yet, comparatively little is known about the influence of surface structures beneficial for biofilms as production platforms ^2,3. This understanding is indispensable to establish stable and highly productive biofilm processes. In this study, the model organism Lactococcus lactis subsp. lactis was used, which produces the antimicrobial peptide nisin (E234). Even though its potential for clinical use has been recognized over the past two decades and the application extended to biomedical fields, its widespread use is restricted due to high production costs and relatively low yields ⁴. Within this study, microstructured metallic substrata were investigated. All surface structures were characterized via optical profilometry and L. lactis biofilms were cultivated in custom built flow cells. Biofilm morphology was analyzed via optical coherence tomography (OCT) and qRT-PCR was used to analyze relative gene expression levels of nisin genes. Biofilm thickness as well as mushroom count varied depending on the substratum used. This morphological dependency on the surface structure rather than solely on fluid dynamics was demonstrated with a hybrid substratum which was only partly structured. Two separate and morphologically distinct sections were further investigated in order to identify structure-based variations in gene expression. Increased gene expression levels were detected for all genes investigated in the sample of the mushroom rich biofilm section. For the structural gene nisA and nisP, a gene involved in nisin processing, particularly high levels were detected. This indicates an increased activity of the entire nisin gene cluster. Even though mRNA levels cannot directly be linked to respective product titers, it is rather interesting to see different behaviors of biofilm sections on the transcriptional level. In addition to the influence of the substratum surface on biofilm morphology, this knowledge can be used to design biofilm processes based on beneficial surface structures.

The financial support by DFG - Collaborative Research Center 926 (Microscale Morphology of Component Surfaces) is gratefully acknowledged.

How to cite: Kleine, D., Breuninger, P., Maus, A.-L., Antonyuk, S., and Ulber, R.: Structural differences of biofilms, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-109, https://doi.org/10.5194/biofilms9-109, 2020.