Biofilms最新文献

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

Effective biofilm disinfection is difficult to be implemented in healthcare settings and industry. In particular, surface disinfection is crucial to prevent microbial contaminations. However, disinfectants misuse has led to an increased concern on the existence of resistance and cross-resistance phenomena due to inadequate disinfection practices. The purpose of this study was the development of a formulation to be used for surface disinfection with wipes. The idea was to produce a formulation based on the combination between the quaternary ammonium compound - cetyltrimethylammonium bromide (CTAB) and a natural product - cinnamaldehyde. In addition, a new disc methodology to assess wiping efficiency was developed based on the Wiperator test (E2967-15) and on the quantitative test method for the evaluation of bactericidal and yeasticidal activity on non-porous surfaces with mechanical action employing wipes in the medical area, 4- field test (EN 16615:2015). The combination of CTAB and cinnamaldehyde was synergic in terms of antimicrobial action against Escherichia coli and Staphylococcus aureus. After stablishing the final formulation, wiping efficacy was assessed with the new methodology. In this case, a contaminated surface (6.20 ± 0.21 log₁₀ CFU of E. coli and 7.10 ± 0.06 log₁₀ CFU of S. aureus) was wiped using two different wipes in terms of composition, thickness and porosity (A and B). After wiping the contaminated surface with wipe A, without the formulation, 3.42 ± 0.46 log₁₀ CFU (E. coli) and 5.38 ± 0.20 log₁₀ CFU (S. aureus) remained on the surface while in the presence of the formulation the bacteria present were under the limit of detection for E. coli and 2.76 ± 0.22 log₁₀ CFU for S. aureus. The formulation was also able to prevent the transfer of bacteria to clean surfaces after wiping the contaminated surface. In the case of wipe A, after wiping the contaminated surface and the subsequent 2 clean surfaces, a total reduction of 4.35 ± 0.22 log₁₀ CFU and 4.27 ± 0.22 log₁₀ CFU was achieved when the wipe was impregnated with the formulation in comparison with 2.45 ± 0.41 log₁₀ CFU and 1.50 ± 0.35 log₁₀ CFU of removal just by mechanical action for E. coli and S. aureus, respectively. For wipe B a general lower reduction was observed but the same behaviour was detected with the use of the formulation when comparison to just mechanical action. This work highlights the enormous potential of combinatorial approach to increase the efficacy of already used biocides diminishing their in-use concentration and consequently their environmental and public health burden.

Acknowledgements

This work was financed by: UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE - funded by national funds through th

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.

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.

Erosion resistance is one of the advantages bacteria gain by producing biofilms. While it is undesirable for us humans when biofilms grow on medical devices or industrial pipelines, biofilms with a high erosion resistance can be advantageous for biotechnological applications. Here, we demonstrate how the erosion resistance of B. subtilis NCIB 3610 biofilms can be enhanced by integrating foreign (bio)polymers such as γ-polyglutamate (PGA), alginate and polyethylene glycol (PEG) into the matrix during biofilm growth.

Artificial enrichment of the NCIB 3610 biofilms with these biopolymers causes a significant increase in the erosion resistance by slightly changing the surface topography: A decreased cavity depth on the surface results in an alteration in the mode of surface superhydrophobicity, and we obtain a state that is located somewhere between rose-petal like and lotus-like wetting resistance. Surprisingly, the viscoelastic and microscopic penetration properties of the biofilms are not affected by the artificial incorporation of (bio)polymers. As we obtained similar results with all the biopolymers tested (which differ in terms of charge and molecular weight), this indicates that a variety of different (bio)polymers can be employed for a similar purpose.

The method introduced here may present a promising strategy for engineering beneficial biofilms such, that they become more stable towards shear forces caused by flowing water but, at the same time, remain permeable to nutrients or other molecules.

Butanol and Isopropanol are naturally produced by the bacteria C. beijerinckii. Those products are used in large field of applications such as fuel and bulk chemicals. Since butanol is toxic at small concentration for cells, bacterial growth and metabolism are inhibited during classical batch fermentation (1). These phenomena lead to the production of low solvent concentration (around 7 g.L^-1) and a low volumetric productivity (0,13 g.L^-1.h^-1) (2). Continuous fermentation can be performed in order to avoid product inhibition by  a continuous removal of fermentation broth. However, the solvent productive biomass is easily washout at high dilution rate because of the low maximum growth rate of the strain in this metabolism phase  (0,05 h^-1) (3). To overcome this issue, cell immobilization of  C. beijerinckii by biofilm formation on solid support is the best solution. As a result, the biomass residence time can be uncorrelated from the hydraulic residence time leading to a higher viable biomass concentration in the bioreactor and consequently a higher volumetric productivity (up to 5 g.L^-1.h-1 ) (4). Our study aimed  at evaluating biofilm viability which is an important parameter that is linked to process productivity and has been little studied in the case of the IBE fermentation (5).

In this study we developed two techniques to monitor biofilm viability during immobilized cell fermentation: Flow cytometry (FC) and PMA qPCR. After FC analysis, a high background noise due to the biofilm extra polymeric substance is obtained. Consequently, an enzymatic  sequential enzymatic biofilm deconstruction using Dnase I and Proteinase K was developed . This pre-treatment successfully lowered the background noise of this analysis. The suspensions obtained were stained with carboxyfluoresceine diacetate (cFDA) and propidium iodide (PI) which are indicators of cellular activity and alteration of membrane integrity, respectively,  and analyzed by flow cytometry. The percentage of viable cells obtained after pre-treatment compared to the control sample is increased from 2.6 ± 0.9 % to 22.8 ± 8.6% because of the background noise decrease. PMA-qPCR confirmed the results obtained by flow cytometry without using enzymatic pre-treatment. Although FC is less accurate than PMA-qPCR, this technique is less time-consuming, cheaper and reliable to study biofilm viability.

References

1. Jones et al (1986) Acetone-Butanol Fermentation Revisited, Microbiological Reviews 50, 484–524.
2. Ferreira dos Santos Vieira, C., Maugeri Filho, F., Maciel Filho, R., and Pinto Mariano, A. (2019) Isopropanol-butanol-ethanol (IBE) production in repeated-batch cultivation of Clostridium beijerinckii DSM 6423 immobilized on sugarcane bagasse, Fuel, 116708.
3. Ahmed, I., Ross, R. A., Mathur, V. K., and Chesbro, W. R. (1988) Growth rate depend