Orals

Biofilms prevention and removal are crucial in many industrial and medical applications. Their complex and cohesive structure provides resistance to cleaning even to strong disinfectants. A key factor for their behavior is the wetting properties of their surfaces.

The main goal of this work is to study the wetting properties of biofilms produced by bacteria Pseudomonas fluorescens. Biofilms are obtained on glass coupons under well controlled flow conditions, using custom-made flow cell devices. Different nutrient concentration and shear flow conditions are investigated.

Biofilm wetting properties are examined under imposed external body forces (forced wetting) through a specialized device, named Kerberos®. Kerberos® is capable of subjecting sessile droplets to varying tilting angles and centrifugal forces while monitoring the variation of the droplet shape in X, Y and Z-directions through three Wi-Fi cameras. Wetting experiments are carried out using water-based solution (dye solution) droplets on biofilm-coated glass coupons. In this work, spreading/sliding behaviour of droplets are investigated only on horizontal substrates (no tilting) under the action of centrifugal forces. Apart from wetting properties, biofilm growth kinetics and surface morphology at different nutrient and shear flow conditions are also assessed.

Results show that, according to the different growth conditions, biofilms present different wetting properties. At lower nutrient concentration and shear flow conditions, spreading and sliding behaviour are similar to that observed in glass coupons in the absence of biofilm. At higher nutrient and shear flow conditions, spontaneous wicking of the biofilm occurs the moment of droplet deposition on the biofilm leading to irregular and jagged shapes of droplets, while on the contrary water droplets look like smooth spherical sections on pure glass. The spontaneous wicking affects the droplet initial shape and so the wetting behaviour during the subsequent rotation tests. In each examined condition, biofilms show hydrophilic properties.

How to cite: Recupido, F., Petala, M., Kostoglou, M., Caserta, S., Guido, S., and Karapantsios, T. D.: Wetting properties of biofilm-coated surfaces produced at controlled shear flow conditions, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-140, https://doi.org/10.5194/biofilms9-140, 2020.

Fighting microbial biofilms on biomaterials is usually addressed by incorporating antimicrobial agents. Nevertheless, as usual in the natural life, intrinsic properties of the material surface can also be a complementary approach. They may drastically reduce the quantity of adhered microorganisms and the remaining microorganisms can be treated with classical antimicrobial agents. Mechanical properties of material surfaces recently emerged as a possible way to impact biofilm formation, but many questions have still to be elucidated so far.

We have especially investigated whether hydrogel and non-hydrogel soft and stiff films may differently impact, microbial behavior and biofilm formation. The films have been thoroughly characterized in terms of viscoelasticity, hydration and chemistry. Microbial mobility, adhered quantity and production of organelles such as pili have been specifically investigated. Surface properties, especially mechanical ones, have been thoroughly characterized. The study has been conducted with yeast (Candida albicans) and bacteria species (Escherichia coli) as models. Our results reveal that the stiffness differently impacts the amount and mobility of the adhered cells according to the nature of the film.  These softness- and hydration-dependent microbial phenomena also vary with bacteria and yeast species.

Finally, this confirms the relevance of using some soft coatings to prevent biofilm formation on a material but also clarifies the risks to get opposite effects as desired if other crucial surface properties have not been associated.

How to cite: Vigué, A., Vautier, D., Hardouin, J., Arntz, Y., Ball, V., Senger, B., Jouenne, T., and Ploux, L.: Soft coatings, a new antimicrobial strategy for biomaterials?, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-14, https://doi.org/10.5194/biofilms9-14, 2020.

Drinking water distribution systems (DWDS) are an engineered system designed to protect water quality during delivery from treatment works to consumers’ taps. Biofilms form on the vast internal surfaces of DWDS, impacting water quality by their activity and/or mobilisation into the bulk-water. Disinfection-residuals are often maintained in drinking water to mitigate planktonic microbial contamination (and associated water quality/health risks). However, the impact of residual-disinfection upon biofilms, and the subsequent unintended risk they may present to water quality, is unclear.

To address this, an internationally-unique, temperature-controlled, full-scale DWDS test facility, fed with water from the local DWDS, was used to grow biofilms (for 28 days). The facility enables three simultaneous conditions to be run in replicate pipe loops (each ~200m long, 79mm internal diameter, PE100 pipe). Conditions studied were Low-, Medium- and High-chlorine regimes. Various water quality parameters were monitored throughout, biofilms were sampled every two weeks (n=5). Physical, chemical and molecular analyses were applied to characterise the matrix (structure and composition) and microbial communities (via analysis of bacterial 16S rRNA and fungal ITS genes) of biofilms developed under the different chlorine regimes. After growth, a “mobilisation” test was conducted simulating hydraulic changes that occur in DWDS. Biofilms from each chlorine regime were exposed to increasing shear stresses to determine any water quality degradation as a consequence of biofilm mobilisation.

High-chlorine residual concentration during development reduced biofilm bacterial concentrations but increased inorganics and selected for unique bacterial and fungal communities. Ultimately the biofilms developed under a High-chlorine residual resulted in the greatest decrease in water quality, in response to mobilisation, and the Low-chlorine regime resulted in biofilms which had the lowest impact on water quality. These unanticipated findings suggest chlorine-boosting should be considered carefully and may actually exacerbate water quality issues. The derived understanding could impact the long-term management of DWDS water quality and biofilm, whilst challenging the current mind-set of continuous residual-disinfection control strategies.

How to cite: Fish, K., Gaskin, P., and Boxall, J.: Residual-chlorine concentration impacts the ecology of biofilms in drinking water pipes and their water quality response, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-16, https://doi.org/10.5194/biofilms9-16, 2020.

Biofilm development in industrial settings can prove costly to manufacturing and consumer health. The presence of contaminants in raw materials, finished products and on process contact surfaces negatively impacts on product quality and safety. Rapid and accurate identification of spoilage and pathogenic microorganisms is crucial to implement effective biofilm control strategies that enhance product safety. The application of confocal Raman microscopy (CRM) for non-invasive and rapid characterisation of clinical and food isolates has been reported. The question remains whether the technique can be used as an online monitoring tool for real-time measurement of biofilm build-up in dynamic manufacturing conditions.

In this study we investigated if CRM could be used in the manufacturing environment as an alternative microbiological quality control method. We assessed if this technology is able to differentiate between bacterial species and their growth phenotype, as well as detect contaminants from process samples.

Laboratory and industrial isolates grown under different culture conditions (planktonic, agar plates, and CDC grown biofilms), and formulated products were analysed using a confocal Raman microscope (Thermofisher DXR2xi) under optimised settings. Reference and experimental Raman spectra were collected and analysed for all test conditions [1]. Spectral similarities were evaluated by developing a microbial multivariate predictive model using a two-way orthogonal partial least squares (O2PLS) regression for cluster analysis [2].

Optimal spectra for microbes were obtained in the fingerprint region at approximately 600 - 1800 cm^-1 where characteristic peaks could be assigned to different biological macromolecules (nucleic acids, proteins, lipids and carbohydrates). Cluster analysis showed good group separation with low variation within but high variation between bacterial strains, enabling bacterial differentiation. It also highlighted the variations observed in the spectral fingerprint for planktonic, agar and biofilm growth modes. Comparative studies suggest that the peak associated with nucleotide ring stretching (~ 700 cm^-1) could be used as a microbial marker for contamination in formulation.

Our findings indicate that confocal Raman microscopy can be used for at-line monitoring of contamination in product streams. Raman spectra provide biochemical data for microbial characterisation but variations in the spectra are often difficult to observe and interpret. Multivariate statistical methods permit rapid interrogation of spectral data, with the potential to improve microbial identification. In combination with multivariate analysis, CRM can be used as an analytical tool for rapid identification of industrial isolates and differentiation of their growth phenotype.

References

[1] Beier, B.D., R.G. Quivey, and A.J. Berger,Raman microspectroscopy for species identification and mapping within bacterial biofilms.AMB Express, 2012. 2(1): p. 35-35.

[2] Zou, X., et al., Automatic Spectroscopic Data Categorization by Clustering Analysis (ASCLAN): A Data-Driven Approach for Distinguishing Discriminatory Metabolites for Phenotypic Subclasses.Analytical Chemistry, 2016. 88(11): p. 5670-5679.

How to cite: Magalhaes, A., Goldberg Oppenheimer, P., Overton, T., and Wright, K.: Real-Time Monitoring of Industrial Biofilms using Confocal Raman Microscopy and Multivariate Analysis, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-35, https://doi.org/10.5194/biofilms9-35, 2020.

Stenotrophomonas maltophilia is a multidrug resistant human nosocomial opportunistic pathogen. It contributes to disease progression in cystic fibrosis patients and is found in wounds, other infected tissues and on catheter surfaces. S. maltophilia is globally distributed and forms 23 distinct phylogenetic clusters (1, 2). Due to its multidrug resistance, it is extremely difficult to heal S. maltophilia caused infections. Colistin is a last-resort antibiotic against multidrug resistant pathogens. However, this study reveals that the minimal inhibitory concentration (MIC) of colistin varies strongly between 22 tested clinical isolates by ranging from 6.25 - >100 µg/ml. The minimal biofilm inhibitory concentration (MBIC) was detected to be much higher. On 41% of the isolates, colistin proved to be very effective on planktonic cells (MIC-value ≤6.25 µg/ml), but less effective on biofilm cells represented by only 18% of the isolates (MBIC-value <100 µg/ml). Thus, we screened for substances, which prevented specifically the biofilm formation or were involved in the removal of established biofilms. We identified several natural fungal compounds and synthetically produced analogues that affect the biofilm of S. maltophilia. In microtiter plate assays, the three substances HH-R6, HH-R8 and HH-R9, which belong to the rubrolides, had with 63 - 83 % the strongest biofilm reduction effect on the biofilm of S. maltophilia K279a. However, microscopy of the biofilms still revealed some living adhered cells although the biofilm structure was strongly impaired. Furthermore, the antibiofilm effect and the impact on the biofilm structure varied strongly among different clinical S. maltophilia isolates. Ongoing transcriptome analyses are expected to shed light on the biofilm inhibiting mechanism of these substances and to get further evidences how they can be used in a clinical setting in the future.

1   Steinmann J., Mamat U., Abda E.M., et al. Analysis of Phylogenetic Variation of Stenotrophomonas maltophilia Reveals Human-Specific Branches. Front Microbiol. 2018, 9:806 (2018). doi:10.3389/fmicb.2018.00806

2   Gröschel, M.I., Meehan, C.J., Barilar, I. et al. The phylogenetic landscape and nosocomial spread of the multidrug-resistant opportunist Stenotrophomonas maltophilia. Nat Commun 11, 2044 (2020). https://doi.org/10.1038/s41467-020-15123-0

How to cite: Gudzuhn, M., Alio, I., Steinmann, J., Schützenmeister, N., and Streit, W. R.: Substances based on natural fungal compounds inhibit the biofilm of various Stenotrophomonas maltophilia isolates, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-53, https://doi.org/10.5194/biofilms9-53, 2020.

Microbial biofilms are a crucial problem in many areas including the food processing industry, biotechnology, water quality and medical scenarios. The complexity of biofilm formation and subsequent prevention strategies - requires a fundamental understanding of the involved molecular mechanisms and the possibility of long-term monitoring biofilm formation. Infrared attenuated total reflection (IR-ATR) spectroscopy is a versatile analytical technique for monitoring biofilm formation of bacteria isolates in situ, non-destructively, and close to real time as an innovative approach providing molecular insight into biofilm formation [1]. The utility of IR-ATR to investigate microorganism behavior within biofilms derives from the evanescent field penetrating few micrometers into the biofilm formed directly at the interface of a multi-reflection ATR waveguide and the sample. In the present study, isolates from food biogenic amine (BA)-producing bacteria, Lactobacillus parabuchneri DSM 5987 strains formed in cheese are analyzed for developing a deeper understanding on the formation of biofilms, which are significant contributors to the presence of histamine in dairy food products [2]. Infrared spectra were recorded using a custom flow-through ATR assembly for revealing the metabolism of microorganisms within such biofilms along with the effects of the substrate functionality and culture conditions on the extracellular biopolymeric matrices [3,4]. The appearance of key IR bands in the region of 1600-1200 cm^-1 indicates the production of lactic acid or lactate and the presence of amide groups, while most pronounced intensities in 1140-950 cm^-1 correspond to phospholipids, polysaccharides and nucleic acids. In this study, the spectral region between 1700 and 600 cm^-1 was determined to be the representative region for the identification of Lactobacillus parabuchneri biofilms enabling to study bioadhesion mechanisms and physico-chemical property changes during extended periods of biofilm growth. Real time monitoring has led to concrete steps for inhibition and disintegration via suitable antimicrobials by deposition on the IR inactive region of ATR waveguide. Multivariate data evaluation and classification strategies were applied to enable efficient multiparametric analysis for providing molecular information facilitating a better understanding of biofilm formation, maturation and changes in biofilm architecture via IR spectroscopic data. 
 
Keywords: IR-ATR spectroscopy, in situ monitoring, Lactobacillus parabuchneri, biofilm, ATR waveguide, flow-through ATR, lactic acid, multivariate data analysis. 

References: [1] Stenclova P, Freisinger S, et al. Appl. Spectro., 2019; Vol.73 (4) 424-432 [2] Yunda E, Quilès F, et al. Biofouling, 2019; Vol.35 (5) 494-507 [3] Diaz M, del Rio B, et al. Food Microbiol., 2016; Vol.7 (591) 85-91 [4] Lorite G, de Souza A, et al. Colloids Surfaces B. Biointerfaces, 2013; Vol. 102 519-525 

How to cite: Bajrami, D., Kranz, C., Fischer, S., Barth, H., Sportelli, M. C., Cioffi, N., and Mizaikoff, B.: IR-ATR spectroscopy for in situ long-term monitoring of Lactobacillus parabuchneri biofilms , biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-146, https://doi.org/10.5194/biofilms9-146, 2020.

Marine biofilms are assemblages of microbial cells irreversibly attached to living or non-living surfaces, embedded in a self-produced matrix of hydrated extracellular polymeric substances (EPS). The phenomenon of biofouling occurs upon the adhesion and accumulation of biofilms, composing the primary colonizers that are capable of EPS production, followed by the sequential growth of secondary colonizers on submerged structures. Biofouling constitutes a significant issue in marine industries (e.g. maritime transportation) and problems related to biofilm fouling include an increase in drag force, modification of surface properties (e.g. metal corrosion) and production of chemical compounds with inhibition effects to other foulers. The use of powerful biocides exhibits a good performance against biofouling, however, often their efficacy is evident to a lesser degree against biofilms. These chemically active compounds have been found to have toxicity effects for marine life and there is a need to discover high-performance environmentally acceptable products.

The aim of the present study was to investigate the biofilm community composition and gene-expression on commercial antifouling (AF) coatings employing next-generation sequencing approaches. Natural mixed-species biofilms were examined after a four-month immersion of two commercial AF coatings, including a biocidal (BAF) and a fouling release (FR), and a control non-treated surface in Langstone Harbour UK. Replicated biofilm samples were used for nucleic acid extraction and sequenced targeting the 16S rRNA gene and metatranscriptome.

We uncovered distinct biofilm community profiles between the two coatings; the BAF samples were dominated by Bacillariophyceae (diatoms), contrary to the FR and control samples where Oscillatoriophycideae (phylum Cyanobacteria) were prevailing. Alphaproteobacteria and Gammaproteobacteria contributed to a high abundance in all samples. Biofilms on BAF samples exhibited a lower species diversity compared to the FR. Here, we introduce a set of functional genes present across all biofilm-associated communities and highlight the differing gene transcriptional profiles in biocidal treatments. The gene transcriptional analysis uncovered highly enriched genes coding for proteins involved in biofilm regulation and formation. We demonstrated that biocidal-associated biofilms harbor genes that regulate defense mechanisms. Overall, the findings highlight links between differentially expressed protein functions and effects of AF coating type during biofilm development. We anticipate these results to contribute towards further development of antibiofilm strategies and fill gaps related to marine biofilm functions.

How to cite: Papadatou, M., Robson, S., Watts, J., Dobretsov, S., and Salta, M.: Functionality and composition of marine biofilms on antifouling coatings., biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-117, https://doi.org/10.5194/biofilms9-117, 2020.

In many environmental systems, such as membrane filtration systems, biofilm control is essential, but costly and requiring harsh chemicals. More effective biofilm control may be obtained using a “materials science” approach.  Biofilms can be characterized as viscoelastic materials, and biofilm “disruptors” can be characterized for their weakening effect on biofilm mechanical strength. By using a novel mathematical model that incorporates biofilm mechanical properties, fluid flow, and diffusion and reaction of disruptors, better cleaning strategies can be devised.

Phase-field models, where the biofilm is treated like a viscoelastic fluid, are one of the few types of models that can predicting deformation and detachment based on mechanical properties. While several related studies have proposed phase-field models for predicting biofilm deformation, there has not been any validation of these models with experimental data. As a first step towards developing a material science strategy for biofilm control, this study validated the ability of a phase-field model to capture biofilm viscoelastic behavior.

In this study, a two-dimensional continuum biofilm model was implemented with finite element method (FEM) using COMSOL Multiphysics (COMSOL v5.4, Comsol Inc, Burlington, MA). We applied the phase-field model with the Cahn-Hilliard equation to simulate biofilm mechanical behavior under fluid flow. The Oldroyd-B model, the simplest viscoelastic constitutive model, was applied to capture biofilm viscoelasticity. The biofilm was modeled as an incompressible viscoelastic fluid, with EPS and a water solvent. The phase-field physics were adapted from previous studies and applied to biofilm-fluid interactions. Two types of incompressible, immiscible fluids (EPS and water solvent) were studied as two components of a single fluid, with a fluid-fluid interface between the two.

Homogeneous alginate was used as a synthetic biofilm for the experimental validation. The viscoelastic parameters of alginate were obtained by shear rheometry using stress relaxation tests. In experimental tests, the deformation behavior was observed in real time using optical coherence tomography (OCT). By importing the 2-D geometry from OCT and viscoelastic parameters from rheometry, the model was simulated and compared with real deformation in the flow cell.

With the applied constant flow (Re=6), biofilm demonstrated viscoelastic behavior.The same behavior was observed in modeling as well. By tracking the movements of several locations of the biofilm geometry, it was concluded that the deformation of alginate biofilms was consistent with the computational results of phase-field models. The relative error between experiment and model for this certain location were 12.8%. Heterotrophic counter-diffusional biofilms cultured in membrane-aerated biofilm reactors were also tested in this study, with a relative error of 22.2%.

In conclusion, the phase-field model, coupled with Oldroyd-B equation, could properly capture biofilm viscoelastic behavior. In a complex system, the phase-field model could be used as a tool to characterize the viscoelastic parameters from the observed deformation. With this information, the model can be used to predict the required disruptor dose to achieve high amounts of biofilm removal with a minimal amount of chemical addition. This can reduce operating costs and minimize the use of harsh chemicals.

How to cite: Li, M., Matouš, K., and Nerenberg, R.: Prediction of biofilm deformation and detachment using shear rheometry, phase-field modeling, and OCT, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-106, https://doi.org/10.5194/biofilms9-106, 2020.

Many bacteria use repeats-in-toxin (RTX) adhesins to mediate binding to host cells and facilitate subsequent colonisation and infection by forming biofilms. Vibrio cholerae, the causative agent of cholera, uses a 230-kDa RTX adhesin, FrhA, to facilitate intestinal colonization. FrhA also mediates hemagglutination of red-blood cells (erythrocytes). Here we have demonstrated that the hemagglutination capability of FrhA is localized to a ~ 20-kDa domain near its C terminus. Bioinformatic analyses indicated this erythrocyte-binding domain (VcEBD) is 65% identical to a peptide-binding module found in the 1.5-MDa ice-binding RTX adhesin that helps its Antarctic bacterium, Marinomonas primoryensis, form symbiotic biofilms with diatoms on the underside of sea ice. This suggested that the FrhA binds V. cholerae to proteins present on the cell surface of erythrocytes. X-ray crystallography revealed that VcEBD has an oblong β-sandwich fold with a shallow, Ca²⁺-dependent ligand-binding cavity that can anchor a peptidyl ligand with a free terminal carboxyl group. Using a structure-guided approach, we screened a small library of ~ 60 short peptides and optimized the affinity of VcEBD’s peptidyl ligands by roughly 1,000-fold. Importantly, the high-affinity ligands are effective at blocking V. cholerae from binding to erythrocytes at nano-molar concentrations. Structures of VcEBD in complex with three different peptides further elucidated the molecular basis for their interactions, which sets the stage for the development of ligand-based antagonists that may help disrupt V. cholerae interaction with intestinal cells to prevent or treat cholera. With the spread of antibiotic-resistant pathogenic bacteria, this work sheds light on an anti-adhesion approach for combating bacterial infections without the excessive use of antibiotics.

How to cite: Guo, S., Lloyd, C., Kinrade, B., Sherik, M., Voets, I., Klose, K., and Davies, P.: Blocking Vibrio cholerae-mediated hemagglutination with short peptide antagonists, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-121, https://doi.org/10.5194/biofilms9-121, 2020.