Biofilms最新文献

Bacteria living in biofilms tolerate much higher antibiotic concentrations compared to planktonic bacteria and can cause chronic infections. Among the most difficult pathogens to treat, Pseudomonas aeruginosa is responsible for many biofilm-related infections and for much of the mortality associated with airway infections in cystic fibrosis. We speculated that there are specific genes responsible for increased antibiotic resistance in biofilms and aimed to identify them in P. aeruginosa. By doing so, a better understanding of biofilm-mediated resistance can be achieved and new bacterial targets can be identified. A P. aeruginosa transposon mutant library was screened to assess the impact on biofilm formation and the biofilm resistance toward antibiotics. Briefly, the biofilm resistance was estimated by following the re-growth of biofilm cells exposed to different concentrations of antibiotics. A few candidates, e. g. the response regulator CbrB, involved in nutrient uptake, have been identified as crucial for biofilm formation and resistance towards antibiotics. Further characterization of these interesting genes has been carried out to explore the underlying mechanism of resistance. Such knowledge can lead to the identification of susceptibility of P. aeruginosa biofilm and help to develop tools to treat persistent infections.

In the last 40 years, bioelectrochemical systems (BESs) have been increasingly discussed within the scope of debates about sustainable energy sources and production of value added chemicals independent of fossil sources. Since the produced current in microbial fuel cells as well as the turnover rates in microbial electrosynthesis cells are dependent on the biocatalysts´ activity, control of the growing biofilm plays a major role in BESs. Moreover, the knowledge about the interplay between biofilm development and electrochemical parameters is crucial for optimizing these sytems.

In the last 3 years, various electroactive biofilms (anodic and cathodic) were cultivated and characterized in a versatile and house made lab-scale flow cell system as well as in a rotating disc biofilm contactor (RDBC). Both systems allow for control of substrate (liquid and gaseous), and nutritional conditions as well as hydrodynamics and other physical parameters. The monitoring of biofilm development was conducted non-invasively by means of optical coherence tomography (OCT). For cathodic biofilms, quantitative analysis of generated 3D OCT data sets revealed a correlation between substratum coverage and measured current density. The increase of substratum coverage led to a decrease of measured current density due to less abiotic redox processes on the cathode surface. A stable current density was achieved when a substratum coverage of 99 % was reached. Furthermore, calculated biofilm accumulation rates could also be correlated with the substratum coverage. The overall biofilm accumulation rate decreased when the substratum was fully covered. Both correlations support the hypothesis that the availability of electrons from the cathode surface is a limiting factor in microbial electrosynthesis.

A 10-liter RDBC was designed to continuously harvest biomass from the electrode to extract intracellularly stored products. In future, this approach could be applied for biotechnological processes. Additionally, the RDBC can be used to obtain reliable mass balances and turnover rates because of its larger scale.

Introduction

Biofilm consists of a complex self-produced matrix of polysaccharides, DNA and proteins that 

protects bacteria from the environment including the host immune system and constitutes the main

cause of bacterial resistance against antibiotics. Research is then focused on finding alternative 

antimicrobial substances able to either hamper biofilm formation or to prevent bacterial growth. 

Recently, we showed that the antimicrobial peptide Temporin-L impairs E.coli growth by inhibiting 

cell division (Di Somma et al.; 2020; BBA). Here we investigate the effect of Temporin-L (TL) on 

biofilm formation in Pseudomonas fluorescens (P. fluorescens) both in static and dynamic conditions, 

showing that TL displays antibiofilm properties. 

Materials and methods

Biofilm formation in static conditions was performed on coverslips and analyzed by the Crystal Violet 

assay. Biofilm morphology was assessed using imaging techniques. Investigation of biofilms in 

dynamic conditions was performed in a flow chamber using a microfluidic system and images were 

recorded by confocal microscopy.

Results

The P. fluorescens cells were either grown in the presence of TL or incubated with the antimicrobial 

peptide after biofilm formation both in static and dynamic conditions using different concentrations 

of the peptide. When TL was added during cell growth, the peptide affected biofilm formation at 25 

µM. Confocal microscopy demonstrated that at this concentration P. fluorescens cells were still alive

but a clear disruption of the biofilm architecture was observed. These results had to be ascribed to a 

specific antibiofilm effect of TL. At 100 µM TL antibiofilm activity biofilm thickness was nearly 

negligible. 

When P. fluorescens cells were treated with TL following biofilm formation, confocal images 

demonstrated that the peptide exerted a strong antibiofilm effect leading to cell detachment and 

disruption the biofilm architecture. 

Discussion and Conclusions 

Investigation of TL effect on P. fluorescens showed that when added during bacterial growth this 

peptide exerted antibiofilm activity at low concentration impairing biofilm formation both in static 

and dynamic conditions, leaving most of bacterial cells still alive. However, confocal microscopy 

measurements could not detect the long necklace-like structures observed in E.coli indicating a 

different mechanism of action of TL on P. fluorescens. Furthermore, when TL was added to a 

preformed P. fluorescens biofilm, the peptide showed a strong antibiofilm activity both in static and 

dynamic conditions, suggesting that TL might penetrate

Biofilms are thought to play a serious role in the food processing environment. Direct contact with or detachment of microorganisms of biofilms could lead to product contamination resulting in diminished shelf life of the product. Packaging and filling are essential key steps for product safety, as any contamination within this step will directly impact the shelf life and safety of the product. For bottled and canned beverages filling lines are used for filling huge amounts of product in a standardised manner. Most parts of these lines are cleaned and disinfected automatically during cleaning in place (CIP) procedures. The design of these filling lines is of great importance, as accessibility and materials are crucial regarding the success of automatic cleaning and disinfection programs. Some companies implemented additional manual cleaning strategies for the complete removal of potential problem-causing sites. In the brewery setting the term biofilm is manifested since the 90s. However, until now all studies focus only on the presence of microorganisms, neglecting the presence of matrix components, which constitutes an essential component of a biofilm.  

Within this study a filling line for cans, capable of filling 60000 cans per hour (volume 0.5 l), was investigated regarding critical sites for biofilm formation. The filling line is used primarily for beer, but mixed beer drinks are also filled. We sampled 23 sites using a scraper-flocked-swab method at two time points. The first sampling was done during operation and the second sampling was conducted one month later after the automated cleaning and disinfection procedure. The samples were characterised regarding their microbial load (qPCR for 16S rRNA and 18S rRNA genes) and the presence of biofilm matrix (phenol-sulfuric assay for carbohydrates, precipitation and SDS-PAGE with subsequent silver staining for proteins and precipitation and spectrophotometric quantitative measurements for extracellular DNA).

During operation, we could identify three sites harbouring a biofilm by applying the definition of presence of microorganisms and at least two matrix components. Furthermore, there were seven sites harbouring microorganisms and one matrix component. After cleaning and disinfection no biofilm could be detected. At one site, microorganisms and one matrix component could be detected. The drastic reduction of biofilm positive sites indicates the successful removal of biofilms by the cleaning and disinfection process.

The design of future filling plants should emphasise on the principles of hygienic design, as this can help to prevent biofilm formation and targeted removal of biofilms during cleaning and disinfection. The here identified biofilm hotspots indicate potential problem-causing sites and weak-points in the design of the filling line.

1. S. I. Hossain^1,3,*, M. C. Sportelli^1,2,3, R. A. Picca^1,3, N. Ditaranto ^1,3, N. Cioffi^1,3

¹Dipartimento di Chimica, Università degli Studi di Bari “Aldo Moro”, Bari, Italy; ²CNR, Istituto di Fotonica e Nanotecnologie UOS, Bari, Italy;       ³CSGI (Center for Colloid and Surface Science) c/o Dept. Chemistry, via Orabona 4, 70125 Bari, Italy.

Copper nanoparticles (CuNPs) are considered as potential antimicrobial agents due to their improved stability and safety, and longer active period than that of organic nanomaterials, with multi-targeted mechanism of action [1]. Nevertheless, metal NPs can suffer from agglomeration, reducing their antibacterial activity [2]. Cu incorporation in inorganic substrates such as metal oxides or montmorillonite (MMT) plays an important role due to the possibilities of creating an antibacterial nanomaterial with slow release of Cu species in order to obtain a prolonged antibacterial activity. Therefore, CuNPs were synthesized via a rapid electrochemical method using the inorganic micro-powders as carrier. Characterization studies on the nanocomposite were done by Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS). The as-prepared Cu-based nanocomposites could be employed for inhibiting the growth of biofilms.

References

1. Nanotechnology 25, (2014), 135101
2. ACS Appl. Mater. Interfaces 4, (2012), 178–184

Acknowledgements

"Financial support is acknowledged from European Union’s 2020 research 
 and innovation program under the Marie Sklodowska-Curie Grant 
 Agreement No. 813439."

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.