bluuebunny/crossref_metadata_2025 huggingface dataset:
The susceptibility of E. faecalis biofilm against selected new quaternary ammonium compounds
Authors: Hympanova Michaela , Terlep Sasa , Markova Aneta , Prchal Lukas , Dogsa Iztok , Marek Jan , Stopar David
Published: July 2020

Introduction: An increasing microbial resistance to known antibiotics raises the demand for new antimicrobials. New antibacterial agent should have a good activity against planktonic as well as biofilm bacteria. Quaternary ammonium compounds (QACs), are widely used in medicine, have proven antimicrobial properties, and are low toxic and low irritating. In this study new QACs were evaluated for their biofilm eradication efficiency as antibacterial compounds and as irrigants in combination with Er:YAG photoacoustic streaming.

Aims: To evaluate the effectiveness of new QACs against E. faecalis biofilms and to increase the effectiveness of QACs with laser treatment.

Method: The biofilm of E. faecalis were grown on titanium surface. The fraction of the dead cells and the biofilm surface coverage was determined with LIVE/DEADTM using confocal microscopy (CLSM) before and after QACs treatment. To enhance to effectiveness the biofilm samples were pretreated with QACs followed by short laser Er:YAG photoacoustic streaming treatment.

Results: All tested QACs were effective against the E. faecalis biofilms. The best anti-biofilm compounds were N-Alkylimidazolinium derivatives. Compared to planktonic bacteria the bacteria in the biofilm were up to 10 fold more resistant. The fraction of the dead bacteria that were treated with QACs followed by Er:YAG photoacoustic streaming increased significantly compared to the chemical treatment alone. In addition, the biofilm surface coverage decreased after laser treatment.

Conclusions: The results suggest that new QACs have a great potential as antibacterial compounds effective against biofilms of E. faecalis. The laser treatment can significantly improve the effectiveness of QACs treatment.
Morphological and diffusional changes in L. lactis biofilms
Authors: Chodorski Jonas , Hauth Jan , Wirsen Andreas , Ulber Roland
Published: July 2020

Through their special way of life, biofilms have several advantages over organisms in planktonic growth. By being surface-attached and producing a mass of extracellular polymeric substances (EPS), microorganisms possess inherent self-immobilization, which decreases the expenditure of downstream processing in industrial applications. Furthermore, they are more resilient against environmental stress and toxic substances, such as antibiotics. An important factor here is diffusion, of substrate into the biofilm and metabolites through and out of the biofilm; however, these mechanisms are still poorly understood. By utilizing a specially developed diffusion model and CLSM FRAP microscopy, diffusion constants in the living, fully hydrated biofilm of L. lactis during development can be assessed. With it, heatmaps of diffusional constants and finally a diffusion profile encompassing a true 3D space of the living biofilm in growth can be generated. With those, possible hotspots and changes of diffusion inside the biofilm structure with regard to changing cultivation conditions and the substratum can be identified, thus furthering our understanding of diffusion in biofilms and how they react to their environment.

The project is funded by the DFG (UL 170/14-1) and the collaborative research center (SFB) 926 on “microscale morphology of component surfaces” (MICOS).
Extracellular DNA and Outer Membrane Vesicles contribute to P. fluorescens SBW25 air-liquid interface biofilms.
Authors: Moshynets Olena , Kayumov Airat , Iungin Olga , Rymar Svitlana , Pokholenko Ianina , Spiers Andrew
Published: July 2020

Outer membrane vesicles (OMVs) and extracellular DNA (eDNA) are important for biofilm formation for many bacteria. OMVs are a perfect transport system to deliver biofilm-related components including eDNA beyond the boundaries of cells, and eDNA itself is an important structural component of biofilms as well as enabling horizontal gene transfer and local adaptation. Both OMVs and eDNA are found in the biofilms produced by the opportunistic human pathogen P. aeruginosa and the plant pathogen P. syringae, but as yet, they have not been reported in the cellulose matrix-based biofilms produced by the related model rhizosphere bacterium Pseudomonas fluorescens SBW25.

In this work we have gone back to re-assess the complexity of SBW25 biofilms by looking for evidence of OMVs and eDNA associated with biofilm–formation. OMVs were first imaged by SEM and LC-MC analysis used to identify 51 biofilm matrix-associated proteins of which 12 were also identified in biofilm OMVs. Interestingly, only 5 proteins were identified in both biofilm matrix and OMV samples, but not in planktonic OMVs, suggesting that these may be biofilm-specific components.  

We also observed eDNA by CLSM in both the weak and poorly-attached Viscous Mass (VM) and robust and well-attached Wrinkly Spreader (WS) air-liquid (A-L) interface biofilms produced by wild-type SBW25 and the Wrinkly Spreader mutant. The eDNA fraction could be precipitated from biofilm cell-free supernatant samples which demonstrated that WS biofilms had two-fold–higher levels than VM biofilms. DNAse treatment effected the development of both types of biofilm and reduced the strength and attachment levels when added to mature VM and WS biofilms. Testing with exogenous DNA suggests that high molecular weight (HMW) DNA is involved in both strength and attachment, perhaps by surface conditioning and interactions with the primary cellulose matrix common to both biofilms. HMW eDNA could be isolated directly from biofilm supernatants whereas two different HMW size fractions could be isolated from OMVs, presumably, from the outer OMV surface because DNAse treatment led to a substantially reduced DNA signal. This suggest that eDNA persistence and degradation in SBW25 biofilms is complex and eDNA fractions may play different roles in biofilm development, protection and adaptation.
The versatile effect of L- and D-Cateslytin on bacteria and yeast biofilms according to configuration, medium and dose
Authors: Ploux Lydie , Jin Min , Hellé Sophie , Betscha Cosette , Strub Jean-Marc , Metz-Boutigue Marie-Hélène
Published: July 2020

L- and D-Cateslytin (CTL) are antimicrobial peptides (AMP) derived from chromogranin A, a protein of the stress response system. Their antimicrobial properties have been thoroughly characterized and already exploited in biomaterials. However, effects on biofilms of yeast and bacteria have never been specifically addressed. We have investigated the impact of both L and D configurations of CTL on the growth of biofilms formed by Candida albicans, Escherichia coli or Staphylococcus aureus microorganisms.

The study was conducted in different media and two strategies of treatment were tested, consisting of administrating the peptide either just at the beginning of biofilm development i.e. on just adhering pioneer microbial cells or on a biofilm already allowed to develop for 24h. We also considered whether the peptide was modified in contact with the medium or/and microbial metabolites. Planktonic and sessile populations of microbial cells were analyzed by spectrophotometry, crystal violet staining, MTT and confocal microscopy with staining by Syto9Ò and propidium iodide. Identification of the peptides and their derived fragments was investigated by HPLC and Mass-Spectroscopy.

In general, CTL-D exhibited higher antibiofilm performances than CTL-L. In addition, concentrations necessary to inhibit biofilm formation were found to vary from ten to eighty times the MICs determined in planktonic cultures. Nevertheless, the results also demonstrate that sessile microorganisms and biofilms are sensitive to CTL (L and D conformations) differently that planktonic populations. Significant (p-value < 0.01) effects were observed on both sessile and planktonic populations and with both strategies of treatments, but they highly varied with medium, species and CTL configuration. Typically, better antibiofilm effect than common antibiotics was reached in some specific conditions, while enhancement of aggregation or biofilm formation occurred in another medium and for other doses. Nevertheless,

Finally, this confirms the quality of CTL peptides as new antimicrobial agents and reveals their anti-biofilm properties. This also specifies the conditions of use necessary to benefit of the highest performances.
CRISPRi enables studies of enterococcal biofilm initiation, maturation and maintenance.
Authors: Afonina Irina , Lu Timothy , Kline Kimberly
Published: July 2020

Among the Enterococci, Enterococcus faecalis is most frequently associated with human infections ranging from the urinary tract and wound infection to endocarditis and bacteraemia. These infections are often multidrug-resistant and, hence, life-threatening. Moreover, E. faecalis are often co-isolated with other pathogenic bacteria from polymicrobial biofilm-associated infections contributing to disease progression and poorer patient outcomes. Genetic tools to dissect complex interactions in biofilms and mixed microbial communities are largely limited to transposon mutagenesis and traditional allelic exchange methods requiring time- and labour-intensive two-step integration and excision screening that can take a week or more to make a single mutant. We built upon the well-characterized CRISPR interference system using streptococcal dCas9 to develop an easily-modifiable, inducible system for E. faecalis that can efficiently silence single and multiple genes in a matter of hours. We show that this system can silence genes involved in biofilm formation, antibiotic resistance, and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance, and can be used to perturb pre-formed biofilms. This inducible CRISPRi system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal regulation networks within the biofilms, including polymicrobial biofilms. 
Chemical stress contribution in bacterial biofilm formation
Authors: Iungin Olga , Spiers Andrew
Published: July 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.
Influence of essential oils on the biofilm formation and cell agglomeration of Burkholderia cepacia from industrial environment
Authors: Huth-Herms Katrin , Kintzel Alexander , Brehmer Annika , Hein Christoph , Uhlmann Prof. Dr. h. c. Dr.-Ing. Eckart
Published: July 2020

Burkholderia cepacia (B. cepacia) is one of nine species the Burkholderia cepacia complex, a group of gram-negative, motile, non-spore-forming and rod-shaped bacteria. Contamination by B. cepacia is found in different industrial issues. B. cepacia affect manufacturing process chains by contaminating the working fluids with planktonic cells and biofilms. Because of the opportunistic pathogenicity to plants, animals, humans and and the multi-drug resistance, B. cepacia is difficult to treat. An alternative treatment method could be the use of herbal raw materials, such as essential oils and their active ingredients. This study aims: (i) to identify the antimicrobial potential of essential oils on the growth of four B. cepacia isolates, (ii) to analyse the influence of active ingredients, on planktonic growth and biofilm formation, (iii) to better understand the impact of commercial and naturally biocides to cell agglomeration as a precursor to mature biofilms. Starting with agar dilution method to evaluate the antimicrobial potential of twenty-three essential oils against B. cepacia (Burk_09, Burk_23, Burk_52 and Burk_309) isolated from cathodic dip coating systems and the wild type (DSM_7288), it was all ready possible to identify eight essential oils that inhibit the growth of B. cepacia. Serial microdilution was used to determine the minimal inhibitory concentration (MIC) of the essential oils for growth and biofilm formation inhibition of B. cepacia. The MIC of Melaleuca alternifolia and Citrus aurantium dulcis essential oils were tested equally for all strains. Essential oils contain active ingredients against the growth of multi-drug resistant and pathogenic bacteria. From twelve active substances among others, Terpinen-4-ol and Geraniol were identified that inhibited growth and biofilm formation. It is concluded that essential oils and active ingredients have a good antimicrobial potential, demonstrating a possible more environmental-friendly alternative to commercial biocides applying in industrial fluids.
Heterogeneous flow environments mediate defector exclusion during yeast floc formation
Authors: Belpaire Tom , Pešek Jiří , Ramon Herman , Steenackers Hans , Smeets Bart
Published: July 2020

Flocculation of Saccharomyces cerevisiae is cooperative phenotype that offers protection against various external stresses. It is modulated by cell-surface proteins, called flocculins, that bind with sugar residues of neighboring cells, effectively increasing cell-cell adhesion. These flocculins are predominantly encoded by the FLO1, which is considered as a ‘green beard’ gene as it governs both the cooperative phenotype and kin recognition of other cooperators. Conversely, defecting cells, lacking FLO1 expression, can still be incorporated in the flocs and ‘cheat’ on the cooperative benefits.

In this work, we investigate various pathways involved in floc formation and specifically their effect on the exclusion of defecting cells. Initially, we experimentally confirmed the heterophilic binding mode of FLO1 and consequently the potential to ‘cheat’. These measured cell mechanical properties were adopted to calibrate the parameters of an individual cell-based model of interacting yeast cells. Using this model, we investigate exclusion of defecting cells for the two mechanisms of group formation; clonal development (staying together) and shear-driven aggregation (coming together). Our results indicate the need for a multi-stage mechanism and selection for the macroscopic flocs with high degree of defector exclusion, as observed experimentally. The stages in this process are (i) nucleation of clusters with FLO1+ rich cores, either due to aggregation or growth, (ii) high shear flow conditions causing erosion of flo1- cells from clusters and exclusion of aggregates due to remodeling, (iii) fast clustering of small but dense flocs of FLO1+ cells in low shear conditions or due to gravitational sedimentation.
Optimization of PolyHydroxyAlkanoate Bioelectrosynthesis by the thermophilic bacterium Kyrpidia spormannii
Authors: Pillot Guillaume , Sunny Soniya , Comes Victoria , Kerzenmacher Sven
Published: July 2020

The electrosynthesis of valuable compounds by biofilms on electrodes is intensively studied since few years. However, the actual biofilms growing so far on cathode produce mainly small inexpensive compounds such as acetate or ethanol. A novel Knallgas bacteria, Kyrpidia spormannii have been recently described to grow on cathode in thermophilic and microaerophilic conditions, producing significant amount of PolyHydroxyAlkanoates (PHAs) (Reiner et al., 2018). These PHA are promising sustainable bioplastic polymers with the potential to replace petroleum-derived plastics in a variety of applications. However, the effect of culture conditions and electrode properties on the growth of K. spormannii biofilm and PHA production is still unclear.

We present in this study the successful development and operation of autotrophic biocathode whereby the electroactive biofilm was able to grow by utilizing CO2 and a cathode as the sole carbon and electron source, respectively. We report for the first time, the effect of operating conditions of the Bioelectrochemical system (BES), cathode materials and cathode surface modification on current consumption, biofilm formation, PHA productivity and overall coulombic efficiency of a K. spormannii culture growing on electrodes. In particular, the focus of this study lies on optimization of three main operating conditions, which are the applied cathode potential, pH buffer and the oxygen concentration in the feed gas. Increased biofilm formation and PHA production was observed at an applied potential of -844mV vs. SCE, pH 6.5, O2 saturation of 2.5%, and for a graphite cathode modified by CO2 activation. The PHA concentration in the biofilm reached a maximum of ≈40 μg·cm-2 after optimization. The resultant PHA yield reported after optimization is increased by 12.2 times in comparison to previous results. In conclusion, these findings take microbial electrosynthesis of PHA a step forward towards practical implementation.
Inhibitory effect of microstructured PVDF surfaces on the microbial attachment of respiratory and oral biofilm forming microorganisms
Authors: Uhlmann Prof. Dr. h. c. Dr.-Ing. Eckart , Hein Christoph , Brehmer Annika , Huth-Herms Katrin
Published: July 2020

Bacterial resistance to conventional antibiotics combined with the increasing awareness of the essential role of biofilms in nosocomial infections caused by medical devices has led to a growing interest in new antimicrobial strategies. Since the formation of bacterial resistances represents a permanent risk in the drug treatment of biofilms, the optimisation of surface properties to avoid microbial attachment is gaining further attention. Besides the haematological field, especially in the respiratory and oral sectors, biofilm-forming microorganisms cause major problems. Due to microbial attachment being mainly determined by the surface properties of the respective substrate material, the medically established polymer PVDF was provided with different microstructures in the size s of 1 µm ≤ s ≤ 200 µm in order to influence the wettability. These structures were applied to injection moulding tools by high and ultra precision milling, electrical discharge machining as well as laser machining. The injection moulded, microstructured PVDF samples showed pyramidal, cup-shaped, channel-shaped and random structures in the micrometer range and led to contact angles Ɵ in the range of 50° ≤ Ɵ ≤ 110°. These samples were then tested for their influence on bacterial attachment by typical representatives of haematologic as well as respiratory and oral biofilm formers Pseudomonas stutzeri and Streptococcus salivarius. The microbial growth and the formed biofilms were analysed after 24 h and 72 h via crystal violet staining and fluorescence microscopy. In comparison to unstructured surfaces, a significant reduction of bacterial attachment was found, which correlated with the respective contact angle and surface roughness, the microgeometry of the structures and the cell morphology of the tested microorganisms. Especially the laser structured, channel-shaped surface showed a highly reduced biofilm formation for both strains. The results offer great potential for the reduction of biofilm formation on medical devices. This technology can also be used in the water treatment sector, such as pipe linings, filter surfaces and sensor housings. The economic large-scale implementation of these microstructures requires further research.
Comparative study of chromatographic methods for the analysis of exopolysaccharides from archaeal biofilms
Authors: Meyer Martin , Kuschmierz Laura , Siebers Bettina , Wingender Jost , Schmitz Oliver J.
Published: July 2020

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.

 

Literature:

[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.
Using elastomeric materials to model biofilm physico-mechanical properties and the associated drag penalty
Authors: Jackson Alexandra , An ShiQi , Dennington Simon , Stoodley Paul , Wharton Julian , Longyear Jennifer , Webb Jeremy
Published: July 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.
Recombinant Protein Production and Plasmid Stability in Escherichia coli Biofilms
Authors: Gomes Luciana C. , Monteiro Gabriel A. , Mergulhão Filipe J.
Published: July 2020

Escherichia coli biofilms have a great biotechnological potential since this organism has been one of the preferred hosts for recombinant protein production for the past decades and it has been successfully used in metabolic engineering for the production of high-value compounds.

In a previous study, we have demonstrated that the non-induced enhanced green fluorescent protein (eGFP) expression from E. coli biofilm cells was 30-fold higher than in the planktonic state without any optimization of cultivation parameters [1]. The aim of the present work was to evaluate the effect of chemical induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) on the expression of eGFP by planktonic and biofilm cells of E. coli JM109(DE3) transformed with a plasmid containing a T7 promoter.

It was shown that induction negatively affected the growth and viability of planktonic cultures, and eGFP production did not increase. Recombinant protein production was not limited by gene dosage or by transcriptional activity. Results suggest that plasmid maintenance at high copy number imposes a metabolic burden that precludes high level expression of the recombinant protein. In biofilm cells, the inducer avoided the overall decrease in the amount of expressed eGFP, although this was not correlated with the gene dosage. Higher specific production levels were always attained with biofilm cells and it seems that while induction of biofilm cells shifts their metabolism towards the maintenance of recombinant protein concentration, in planktonic cells the cellular resources are directed towards plasmid replication and growth [2].

It is expected that this work will be of great value to elucidate the mechanisms of induction on recombinant protein production, especially in biofilm cells which have shown potential to be used as protein factories.

 

 

References:

[1] Gomes, L.C., & Mergulhão, F.J. (2017) Heterologous protein production in Escherichia coli biofilms: A non-conventional form of high cell density cultivation. Process Biochemistry, 57, 1-8. https://doi.org/10.1016/j.procbio.2017.03.018

[2] Gomes, L., Monteiro, G., & Mergulhão, F. (2020). The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by Escherichia coli Biofilms. International Journal of Molecular Sciences, 21(2), 576. https://doi.org/10.3390/ijms21020576
Study of Biofilm Growth on Slippery Liquid-Infused Porous Surfaces Made from Fluoropor
Authors: Keller Nico , Bruchmann Julia , Sollich Thomas , Richter Christiane , Thelen Richard , Sachsenheimer Kai , Kotz Frederik , Schwartz Thomas , Helmer Dorothea , Rapp Bastian E.
Published: July 2020

Avoiding undesired growth of biofilm is a fundamental challenge for all surfaces in long-term contact with aqueous media. Slippery liquid infused porous substrates (SLIPS) are a promising type of surface for preventing biofilm attachment. The effectiveness of SLIPS is based on the liquid/liquid interface between the medium and the surface, which prevents biofilm attachment. However, the long-term stability of these surfaces is problematic: under shear force, the oil layer is removed and the repellent effect is lost. Here, we study correlations between the porosity of the infused substrate and the ability to uphold the SLIPS oil-film under low shear and high shear force conditions. For this purpose, we manufacture substrates with different porosity and surface roughness in porous fluorinated polymer “Fluoropor”, which we have recently introduced. The porous layers were infused with fluorinated oil and their roughness was studied by white light interferometry. We find that SLIPS samples with smaller pores more effectively reduce Pseudomonas aeruginosa biofilm growth in a seven-day microfluidic flow cell experiment. With its easy production, simple adjustment of porosity and the possibility to attach the polymer to various technical substrates during polymerization, Fluoropor is a very promising material for producing stable SLIPS. When produced with small pores, Fluoropor is also transparent and enables the real-time observation of biofilm growth by optical examination. Thus, Fluoropor SLIPS provides an easy approach to reduce bacteria adhesion and bio fouling in many technical applications.
Bioinspired surfaces to delay biofilm formation: different surfaces with different mechanisms
Authors: Chen Jinju
Published: July 2020

Biofilm associated infections are the fourth leading cause of death worldwide, within the U.S. about 2 million annual cases lead to more than $5 billion USD in added medical costs per annum. Therefore, it is important to control biofilm growth and reduce the instances of infections.  Physical strategies, in particular the use of rationally designed surface topographies or surface energies, have present us with an interesting approach to prevent bacterial adherence and biofilm growth without the requirement for antimicrobials.

A variety of natural surfaces exhibit antibacterial properties. Examples of such surfaces include rose petals with hierarchical structures and Nepenthes pitcher plants with slippery liquid-infused porous surfaces.  

In this study, we fabricated different  biomimetic surfaces (rose-petal surfaces and slippery liquid-infused porous surfaces).   We have demonstrated that rose-petal surface can delay early stage P. aeruginosa and S. epidermidis biofilms formation (2 days) by about 70% and control  biofilm  formation according to surface structures.  The mechanisms of hierarchical structures  of rose-petal influence biofilm formation are two folds: 1) Papillae microstructure block  the bacterial clusters in between the valleys, limiting the potential for cell-cell communication via fibrous networks, thereby resulting in impaired biofilm growth. 2) The secondary structure (nano-folds) on microstructures can align bacterial cells within the constrained grooves, thereby delaying cell clusters formation during short term growth of biofilm.

While, the slippery liquid-infused porous surface(s) can prevent over 90% P. aeruginosa and S. epidermidis biofilms formation for a duration of 6 days.  These are mainly attributed to their high contact angle and extreme low contact angle hysteresis.
The role of divalent ions in cariogenic biofilm formation
Authors: Astasov-Frauenhoffer Monika , Steiger Elena , Muelli Julia , Braissant Olivier , Waltimo Tuomas
Published: July 2020

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.
Screening of fast biofilm formation on stainless steel by thermophilic sporeformers originated from dairy powder and their resistance against CIP
Authors: Delaunay Louis , Postollec Florence , Leguerinel Ivan , Mathot Anne-Gabrielle
Published: July 2020

Introduction:

Thermophilic sporeformers are present in raw milk at very low concentration and resist to pasteurisation applicated to destruct vegetative and pathogenic cells. Those spores can adhere to stainless steel due to their hydrophobicity and can form biofilms. Early stage biofilms are important because it can increase the matrix and the adhesion of other cells. Because of those biofilms, the three main species: Geobacillus stearothermophilus, Anoxybacillus flavithermus and Bacillus licheniformis can resists to Cleaning In Place (CIP) procedure, and contaminate a new process.

 

Material and Methods:

Early stage adhesion was conducted on stainless steel submerged by milk inoculated with a fresh culture of bacteria (G. stearothermophilus (N=15), A. flavithermus (N=32) and B. licheniformis (N=15)) for 6h of growth at 55°C under agitation. The ability of sporeformers to form biofilms under those conditions were measured by image analysis after a fluorescent coloration (acridine orange) and random photography. A coverage percentage was calculated by ImageJ ; and a positive threshold was set up at 5% of covering.

The efficiency of CIP procedures were obtained after a caustic soda and nitric acid treatment during different duration and temperature of treatment. Tested biofilms were formed in milk during 12h at 55°C, in stainless steel microplates (96 wells) on the same species (3 strains for each) under agitation. Surviving spores were enumerated by the microcolony method.

 

Results:

Early stage adhesion shows that 62.5 % (N=20) of A. flavithermus strains can form biofilm within 6h, whereas only 6.7% (N=1) of G. stearothermophilus and 0% (N=0) of B. licheniformis biofilm in 6h at 55°C on submerged stainless steel. However, the maximum covering % on A. flavithermus was 35%; while on the only biofilm forming strain of G. stearothermophilus, this percentage reach 75%. Image analysis also shows biofilm structure from 2D to 3D.

The presence and the resistance of spores to chemical cleaning was highly variable within strains. Nitric acid appears to be more effective than caustic soda against biofilms formed by vegetative cells and spores from these strains.

 

Significance:

Those results shows that strong biofilms are mainly composed of spore and are very resistant to CIP used in dairy industries. That is why a better understanding of control methods can lead to a finer and suitableness use of cleaning products.
Phosphodiesterase activity of NbdA from Pseudomonas aeruginosa
Authors: Scherhag Anna , Rüger Martina , Gerbracht Katrin , Rehner Jaqueline , Zehner Susanne , Frankenberg-Dinkel Nicole
Published: July 2020

The molecule c-di-GMP is a bacterial second messenger that controls various processes such as motility or biofilm formation in bacteria [1]. To synthesize and degrade c-di-GMP, enzymes called diguanylate cyclases (DGC) containing a GGDEF-domain and phosphodiesterases (PDE) containing an EAL-domain or HD-GYP-domain are important [1, 2]. Pseudomonas aeruginosa, a model organism for biofilm formation and dispersion, encodes for 18 GGDEF, 5 EAL, 16 GGDEF / EAL, and 3 HD-GYP-domain-containing proteins [3].
One of the GGDEF / EAL-containing proteins is NbdA. This protein also harbors an N-terminal membrane anchored MHYT-domain, that is predicted to be a sensor for NO, CO or O2 [4]. In this work, recombinant and affinity purified NbdA was tested for its PDE activity. Three different methods were used to measure the PDE activity of NbdA: a bis-pNPP-assay in which the conversion of the pseudosubstrate bis-pNPP into p-nitrophenol was detected spectroscopically, an HPLC-analysis of an enzymatic assay with the native substrate c-di-GMP, and a MANT-c-di-GMP-assay in which a fluorescently labeled form of the presumed substrate c-di-GMP was utilized.
To establish these methods, the two known phosphodiesterases, PdeH from Escherichia coli [5] and RocR from P. aeruginosa [6], were also produced and tested. Subsequently, three variants of NbdA were investigated: the full-length version and two truncated versions of the protein. Activity was further assessed using functional complementation of an E. coli phosphodiesterase deficient strain with full-length and truncated NbdA variants confirming PDE activity in vivo.

 

 

[1] Hengge, R. (2009) Nature Rev. Microbiol. 7: 263-273.

[2] Römling, U., Gomelsky, M., Galperin, M.Y. (2005). Mol. Microbiol. 57: 629–639.

[3] Valentini, M., Filloux, A. (2016). J. Biol. Chem. 291: 12547–12555.

[4] Galperin, M.Y., Gaidenko, T.A., Mulkidjanian, A.Y., Nakano, M., und Price, C.W. (2001). FEMS Microbiol. Lett. 205, 17–23.

[5] Pesavento, C., Becker, G., Sommerfeldt, N., Possling, A., Tschowri, N., Mehlis, A., Hengge, R. (2008). Genes Dev. 22: 2434–2446.

[6] Chen et al. (2012) Chen, M.W., Kotaka, M., Vonrhein, C., Bricogne, G., Rao, F., Chuah, M.L.C., Svergun, D., Schneider, G., Liang, Z.-X., Lescar, J.  (2012). Signaling. J. Bacteriol. 194: 4837–4846

 
Biocorrosion research: Are we barking up the right trees?
Authors: Flemming Hans-Curt , Little Brenda , Blackwood Dan , Hinks Jamie , Lauro Federico , Marsili Enrico , Okamoto Akihiro , Rice Scott , Wade Scott
Published: July 2020

Microbially influenced corrosion (MIC), is acknowledged to be the direct cause of catastrophic corrosion failures, with damages ranging to many billions of US$. In spite of extensive research and numerous publications, fundamental questions still remain unanswered. In 1993, J.F.D. Stott published a review paper in Corrosion Science, entitled “What progress in the understanding of microbially influenced corrosion has been made in the last 25 years?“ He concluded, “The most commonly asked question about MIC is: what will be the expected corrosion rate of material x in an environment where aggressive microorganisms proliferate?... For many materials we can no more answer this question now than we could 25 years ago.” Now, over 50 years later, that question is still open. Current MIC research does not provide data related to detection and verification in the field, diagnosing, modelling or prediction. Laboratory experiments seldom attempt to recreate relevant natural or industrial electrolytes. A sober, solution-oriented contemplation of the state-of-art and acknowledgement of the substantial deficiencies in our understanding may help shift MIC research into a direction which could actually produce useful answers.
Extracellular polysaccharides (EPS) secreted by a model marine bacterium (Alteromonas spp.)
Authors: Perez-Cruz Carla , Estupiñan Monica , Llamas-Arriba Mª Goretti , Etxebeste Oier , Lanzen Anders , Alonso-Saez Laura
Published: July 2020

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.
Talk2clean: application of probiotics to control biofilm in industrial water circuits – a innovative application
Authors: KleinJan Hetty , Meunier Christophe , Nonet Stéphane , Michel Mickael
Published: July 2020

Biofilms are omnipresent in industrial cooling water circuits and frequently lead to technical and economic problems. Yet, methods to remove biofilms may be inefficient, due to the EPS layer, which provides a protective layer against penetration of disinfectants. In combination with stricter legislation and increased environmental awareness, this has opened the search for alternative strategies to control biofilms in cooling water systems.

In this study we implement a novel biofilm removal strategy, in which we target the intercellular language involved in the formation of biofilm structures. This language consists of signalling molecules (autoinducers) excreted by biofilm forming bacteria, a process referred to as quorum sensing (QS). We aim to alter QS via the activation of an antagonistic process called quorum quenching (QQ). Quorum quenching is a process that naturally occurs in bacterial communities; hence the final product is environment, - and user-friendly and thus a valuable alternative to the oxidising chemical products that are often used to clean cooling water circuits.

Here we present the first application of a QQ-product tested on pilot scale using parameters that resemble industrial evaporative cooling towers. This setting is particularly interesting as it an open system, fed with various types of make-up water, and comprised of biofilms adjusted to high operating temperatures.

The QQ product was tested using a closed, tube-like system, under continuous flow, fed with a propionic, - and acetic acid rich synthetic medium. Hydraulic retention time (HRT) was gradually shifted over a time span of 6 weeks. Heterotrophic plate counts were acquired once per week from the planktonic and biofilm phase. A pilot without the addition of QQ was ran in parallel as a control.

Our results show that the QQ product reduced and delayed the formation of biofilm compared to the control. Interestingly, this difference diminished when the HRT was modified. Metagenomic analysis of the biofilm phase, revealed that 16S rRNA sequences corresponding to the QQ were also strongly reduced during this shift, indicating a wash-out of the QQ product. The exact interaction of HRT and presence of QQ will be analysed in more depth using QQ specific qPCR primers.
The use of ASH-15 flowstone as a matrix-matched reference material for laser-ablation U-Pb geochronology of calcite
Authors: Nuriel Perach , Wotzlaw Jörn-Frederik , Ovtcharova Maria , Vaks Anton , Stremtan Ciprian , Šala Martin , Roberts Nick M. W. , Kylander-Clark Andrew R. C.
Published: July 2020

Abstract. Latest advances in laser ablation inductively coupled plasma mass spectrometer (LA-ICPMS) allow for accurate in-situ U-Pb dating of carbonate material, with final age uncertainties usually > 3 % 2σ. Cross-laboratory reference materials (RMs) used for sample-bracketing are currently limited to WC1 calcite with an age of 254.4 ± 6.5 (2σ). The minimum uncertainty on any age determination with the LA-ICPMS method is therefore ≥ 2.5 %, and validation by secondary RMs are usually performed on in-house standards. This contribution present a new reference material, ASH-15, a flowstone that is dated here by high-precision Isotope Dilution (ID) TIMS analysis using 36 sub-samples, 1–7 mg each. Age results presented here are slightly younger compared to previous ID dating of ASH-15, but in agreement with in-situ analyses (using WC1 as a RM). We provide new correction parameters to be used as primary or secondary standardization. The suggested 238U/206Pb apparent age, not corrected for disequilibrium and without common-lead anchoring, is 2.965 ± 0.011 Ma (2σ). The new results could improve the propagated uncertainties on the final age with a minimal value of 0.4%, which is approaching the uncertainty of typical ID analysis on, for example, zircon (
New composite bio- and isotope stratigraphies spanning the Middle Eocene Climatic Optimum at tropical ODP Site 865 in the Pacific Ocean
Authors: Edgar Kirsty M. , Bohaty Steven M. , Coxall Helen K. , Bown Paul R. , Batenburg Sietske J. , Lear Caroline H. , Pearson Paul N.
Published: July 2020

Abstract. The Middle Eocene Climatic Optimum (MECO) at ca. 40 Ma is one of the largest of
the transient Eocene global warming events. However, it is relatively poorly
known from tropical settings since few sites span the entirety of the MECO
event and/or host calcareous microfossils, which are the dominant proxy
carrier for palaeoceanographic reconstructions. Ocean Drilling Program (ODP)
Pacific Ocean Site 865 in the low-latitude North Pacific (Allison Guyot) has
the potential to provide a useful tropical MECO reference, but detailed
stratigraphic and chronological constraints needed to evaluate its
completeness were previously lacking. We have addressed this deficit by
generating new high-resolution biostratigraphic, stable isotope, and X-ray
fluorescence (XRF) records spanning the MECO interval (∼38.0–43.0 Ma) in two holes drilled at Site 865. XRF-derived
strontium ∕ calcium (Sr∕Ca) and barium ∕ strontium (Ba∕Sr) ratios and Fe count
records allow correlation between holes and reveal pronounced rhythmicity,
enabling us to develop the first composite section for Holes 865B and 865C
and a preliminary cyclostratigraphy for the MECO. Using this new framework,
the sedimentary record is interpreted to be continuous across the event, as
identified by a pronounced transient benthic foraminiferal δ18O
shift of ∼0.8 ‰. Calcareous microfossil
biostratigraphic events from widely used zonation schemes are recognized,
with generally good agreement between the two holes, highlighting the
robustness of the new composite section and allowing us to identify planktic
foraminiferal Zones E10–E15 and calcareous nannofossil Zones NP15–18.
However, discrepancies in the relative position and ordering of several
primary and secondary bioevents with respect to published schemes are noted.
Specifically, the stratigraphic highest occurrences of planktic foraminifera,
Acarinina bullbrooki, Guembelitrioides nuttalli, and Morozovella aragonensis, and calcareous nannofossils, Chiasmolithus solitus and Sphenolithus furcatolithoides, and the lowest occurrence of
Reticulofenestra reticulata all appear higher in the section than would be predicted relative to other
bioevents. We also note conspicuous reworking of older microfossils (from
planktic foraminiferal Zones E5–E9 and E13) into younger sediments
(planktic foraminiferal Zones E14–15) within our study interval consistent
with reworking above the MECO interval. Regardless of reworking, the
high-quality XRF records enable decimetre-scale correlation between holes
and highlight the potential of Site 865 for constraining tropical
environmental and biotic changes, not just across the MECO but also
throughout the Palaeocene and early-to-middle Eocene interval.