Biofilms最新文献

Colonization of textiles and subsequent metabolic degradation of sweat and sebum components by axillary skin bacteria cause the characteristic sweat malodor and discoloring of dirty clothes. Once inside the textile, the bacteria can form biofilms that are hard to remove by conventional washing. When the biofilm persists after washing, the textiles retain the sweat odor. In addition to posing a huge industrial problem, textile biofilms constitute an interesting case study of bacterial behavior in periodically wetted and dried substrates with varying surface hydrophobicity. Here we aim to study the bacterial behavior in each of the four stages of the bacterial lifecycle in textiles: adhesion, growth, drying and washing. To accomplish this, we designed a novel in vitro model to mimic physiological sweating while wearing cotton and polyester textiles. The hydrophobic polyester adhered bacteria more strongly and absorbed more sebum, the bacteria’s primary nutrient source. Bacteria were therefore initially more active in polyester textiles than in cotton. However, polyester did not bind water as well as cotton. The increased water content of cotton allowed the bacteria to retain a higher activity after the textile had dried. However, neither of the textiles retained enough water upon drying to prevent the bacteria from irreversibly adhering to the textile fibers by capillary action. This demonstrates that bacterial colonization depends on the hydrophobic and hygroscopic properties of the colonized material while highlighting the possibility of controlling bacterial behavior by either changing the surface properties or the surrounding environment.

Nowadays, industrial fermentations rely almost entirely on the use of planktonic cells. However, biofilms (the most common form of bacterial growth in nature), offer several advantages to be exploited in modern fermentation processes. Bacteria in biofilms are more tolerant to several stresses than free cells, including toxic chemicals and shear stress. Furthermore, the adhesion of cells to surfaces can be exploited to operate a continuous fermentation process without excessive loss of biomass, thereby facilitating downstream processing. A programmable switch between planktonic and biofilm lifestyle is desirable to harness the advantages of both lifestyles. On this premise, we constructed a genetic gene circuit for biofilm formation in the platform strains Pseudomonas putida and Pseudomonas taiwanensis. Both P. putida and P. taiwanensis are robust, non-pathogenic soil bacteria and promising chassis for biotechnology as they can thrive under harsh operating conditions, displaying high tolerance towards several chemicals and can metabolize a broad range of substrates. These characteristics make them ideal for the production of a wide spectrum of chemicals. The synthetic circuit initiates biofilm formation upon detection of substrate or intermediate metabolites of the desired biotransformation, thus no additional inducer is needed. The circuit also allows for the propagation of cells in planktonic state prior employment in the bioreactor, which facilitates handling and speed up expansion of the culture. The design proposed herein employs a feedback-resistant diguanylate cyclase (DGC) from Caulobacter crescentus, which increases the concentration of DGC and therefore triggers biofilm formation. The resulting engineered strains were utilized for the biotransformation and degradation of chemicals (cyclohexanol) in continuous cultivation systems. This approach led to a ~300-fold increase in biofilm formation in microtiter plates, and was successfully used in diverse fermentation systems displaying increased catalytic efficiency.

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

            More than 30 years have passed now since the pioneering work of Costerton and co-workers^e.g.,1. We have learned that the biological functions of the cells embedded in the complex, self-produced polymeric extracellular matrix, differ radically from the ones of the planktonic cells. Emergent properties such as enhanced antimicrobial resistance appear.  Biofilms are widely spread in different habitats, both in the environment and the living organisms. Mostly, the characterization of this bacterial specific phenotype has been carried out using mono-species lab models. Yet, these systems are in marked contrast to the biofilms found in the environment. Those are usually complex and contain multiple bacterial species and, in many cases, also fungi, algae, and protozoa². To take this into account, researches have recently turned to multispecies communities, aiming at describing the interspecies interactions in order to decipher the mechanisms underlying the properties of these complex consortia.

            We present here a simplified model community consisting of 4 species — Bacillus thuringiensis, Kocuria salsicia, Pseudomonas fluorescens, Rhodocyclus sp. — elaborated from a natural environment to investigate the mechanisms supporting the formation of a multispecies consortium. We have been able to grow the 4-species biofilm under flow in a millimetric channel made of PDMS, which enabled to monitor the biofilm settlement and development using video-microscopy³. We found a deterministic development which follows defined kinetics and spatial distribution, suggesting that the formation of this adherent community is dominated by the self-induced modulation of the environmental parameters. To clarify this hypothesis, we focused our attention on the spatio-temporal distribution of oxygen and we devised an original experiment to map in situ and in real-time the evolution of oxygen level within the 4-species biofilm.

            We used an O₂ fluorescent probe made of a Ruthenium complex encapsulated in lipidic micelles to overcome the metal toxicity. We derived local oxygen concentration in the biofilm from fluorescent-lifetime imaging microscopy (FLIM) measurements of the probe in situ. The setup was equipped with a light sheet to ensure the optical sectioning for a 3D mapping. We will show here the spatial and temporal characteristics of the method and the first O₂ map obtained on a growing biofilm.

            To conclude, we will discuss how the monitoring of oxygen spatio-temporal distribution in a model community can help to elucidate basic interspecies interactions and reveal general mechanisms likely t

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

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

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

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

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

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

Antibiotic resistance is expected to cause 10 million deaths per year worldwide by 2050. One of the mechanisms for the resilient nature of bacteria toward antibiotics is through the formation of biofilm. Bacterial biofilms are sessile communities of microorganisms, which exist in a matrix of proteins, carbohydrates, eDNA and other various components – collectively known as extracellular polymeric substances. Biofilms slow the penetration of drugs, and also contribute to the development of a resistant phenotype known as persisters. Thus, understanding biofilm composition might contribute to the development of anti-biofilm strategies. The aim of this study was to explore biofilm formed by five Staphylococcus spp ATCC strains, commonly used in research as references: S. aureus 25923, S. aureus 29213, S. aureus 43300 (methicillin-resistant), S. aureus 6538 and S. epidermidis 12228. Biofilm mass and its components were analysed after 24h and 72h of biofilm growth. Bacterial biofilm was prepared in 96-well microtiter plates, in Trypticase Soy Broth supplemented with 1% glucose. After incubation at 37°C, absorbance measurements and crystal violet staining were performed and the specific biofilm formation determined for each strain. Extracellular polymeric substances were extracted using a combination of physical and chemical methods; including centrifugation, vortexing and the use of 1.5M NaCl. In these assays, biofilms were grown in polystyrene tubes containing 10 ml of same media mentioned above. The concentration of protein, carbohydrate and eDNA was determined using the Bicinchoninic acid assay, phenol-sulfuric acid method and DNeasy^® Blood and Tissue Kit, respectively, followed by spectroscopy. Our data demonstrated heterogeneity between the biofilm-forming capabilities and EPS components within staphylococcal strains and species. Strains 25923 and 6538 had the highest value for biofilm formation at both time points. Interestingly, strain 43300 was the only one to show a significant increase in biofilm after 72h. Contradictory to previous findings, S. epidermidis 12228 was found to be a good biofilm producer. At both time points studied, strains demonstrated considerably higher concentrations of protein (varying from 172 µg/mL – 345 µg/mL) and carbohydrate (56 µg/mL - 372µg/mL) in EPS compared to eDNA (2.74 µg/mL – 8.12 µg/mL). On average, strains 43300 and 12228 had the highest concentration of protein, and the latter also had the highest carbohydrate and eDNa amounts at 72h. Strains 25923 and 6538 had a significant decrease in eDNA concentration over time. Based on this brief study, the relative quantities of EPS components investigated is similar to that of other studies with protein being the most plentiful component followed by carbohydrate and then considerably lower amounts of eDNA. Differences in specific bi