Biofilms最新文献

Biofilms are considered as hotspots for the transfer of antibiotic resistance genes (ARGs), but very few studies have investigated the fate of ARGs (e.g. proliferation or elimination) in situ given different microbial spatial self-organization (SSO). SSO refers to a pervasive process during biofilm formation when microbes arrange themselves non-randomly across surfaces. So far the causes of SSO have been uncovered in a sense, however, the consequences of SSO were largely overlooked. Here, I hypothesize that the magnitude of inter-species intermixing, as one fundamental character of SSO, will determine the fate of ARG-carrying conjugative plasmid in both absence and presence of antibiotic selection. I evaluated this by performing range expansion experiments on agar plates to develop an artificial biofilm using a synthetic microbial community consisting of two isogenic Pseudomonas Stutzeri A1501 who are facultative denitrifiers in anaerobic condition. By knocking out different functional genes responsible for different steps of denitrification I am able to modify the metabolic interactions between these two strains from competing (without trophic interaction) to cross-feeding (with trophic interaction), which will further result in different magnitude of inter-species intermixing. Competing group has lower magnitude due to demixing of two, while cross-feeding group has higher magnitude due to mixing. I observed that in the absence of antibiotic selection plasmid experienced faster pace of elimination in competing group than cross-feeding group, whereas in the presence of antibiotic selection plasmid proliferated more efficiently in cross-feeding group than competing group. These results suggest that SSO is a determining factor of the fate of ARGs in biofilms, which provides a novel perspective of better understanding ARGs-related pressing problems facing our society.

Genus Staphylococcus comprises many greatly pathogenic species like S. aureus, S. epidermidis or S. saprophyticus. The great pathogenicity of stated species is often facilitated by their capability to form thick complex biofilms on various biotic or abiotic surfaces. Biofilm formation together with extracellular hydrolases or toxins represents important virulence factor, which increases persistence of staphylococci in host via enhancing their ability to evade host immune system and further promote the infection development. With an increased emergence of antibiotic resistance among pathogenic bacteria including staphylococci the search for novel antibiotic compounds with antivirulence effect is sought. Such substances might be stilbenes, phenolic compounds isolated from various plants (Vitis spp., Vaccinium spp., Pterocarpus spp., Pinus spp.). They possess strong antioxidant activity and a wide spectrum of beneficial pharmacological effects (antitumor, hypolipidemic, hypoglycemic). Apart from that, they also have great antimicrobial activity with a potent ability to enhance antibiotics action in combination.

Presented work focused on resveratrol, pterostilbene (PTE) and pinosylvine and their effect on S. aureus and S. epidermidis biofilm formation. The effect of stilbene representatives on production of other virulence factors (proteases, phospholipases, haemolysins), cell surface hydrophobicity and morphology was also observed.

PTE was found to be the most effective among studied stilbenes against S. aureus and S. epidermidis biofilm with minimum biofilm inhibitory concentrations (MBIC₈₀) ranging from 40 to 130 mg/l. Its effect on mature staphylococcal biofilm eradication was even greater with 80% eradication rate achieved by 40-75 mg/l. PTE (49 mg/l) was found to have a potent combinatory antibiofilm activity with erythromycin or tetracycline (5 mg/l both) causing more than 80% inhibition in metabolic activity of biofilm cells. It was able to permeabilize cytoplasmic membrane, thus probably enabling antibiotic uptake by the cell. PTE also altered cell surface hydrophobicity and production of haemolysin.

PTE might be the solution to increasing biofilm-related resistance problem and a promising candidate with antibiofilm and antivirulence potential for future antibiotic treatment of staphylococcal infections.

This work was supported by the grant of Specific university research – grant No. A2_FPBT_2020_004.

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

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