Graphene-Based Composites for Biomedical Applications: Surface Modification for Enhanced Antimicrobial Activity and Biocompatibility.

The application of graphene-based materials in medicine has led to significant technological breakthroughs. The remarkable properties of these carbon materials and their potential for functionalization with various molecules and compounds make them highly attractive for numerous medical applications. To enhance their functionality and applicability, extensive research has been conducted on surface modification of graphene (GN) and its derivatives, including modifications with antimicrobials, metals, polymers, and natural compounds. This review aims to discuss recent and relevant studies related to advancements in the formulation of graphene composites, addressing their antimicrobial and/or antibiofilm properties and evaluating their biocompatibility, with a primary focus on their biomedical applications. It was concluded that GN surface modification, particularly with compounds intrinsically active against bacteria (e.g., antimicrobial peptides, silver and copper nanomaterials, and chitosan), has resulted in biomaterials with improved antimicrobial performance. Furthermore, the association of GN materials with non-natural polymers provides composites with increased biocompatibility when interfaced with human tissues, although with slightly lower antimicrobial efficacy. However, it is crucial to highlight that while modified GN materials hold huge potential, their widespread use in the medical field is still undergoing research and development. Comprehensive studies on safety, long-term effects, and stability are essential before their adoption in real-world medical scenarios.

Use of Probiotics to Control Biofilm Formation in Food Industries.

Microorganisms tend to adhere to food contact surfaces and form biofilms, which serve as reservoirs for bacteria that can contaminate food. As part of a biofilm, bacteria are protected from the stressful conditions found during food processing and become tolerant to antimicrobials, including traditional chemical sanitisers and disinfectants. Several studies in the food industry have shown that probiotics can prevent attachment and the consequent biofilm formation by spoilage and pathogenic microorganisms. This review discusses the most recent and relevant studies on the effects of probiotics and their metabolites on pre-established biofilms in the food industry. It shows that the use of probiotics is a promising approach to disrupt biofilms formed by a large spectrum of foodborne microorganisms, with Lactiplantibacillus and Lacticaseibacillus being the most tested genera, both in the form of probiotic cells and as sources of cell-free supernatant. The standardisation of anti-biofilm assays for evaluating the potential of probiotics in biofilm control is of extreme importance, enabling more reliable, comparable, and predictable results, thus promoting significant advances in this field.

Development of Cork Biocomposites Enriched with Chitosan Targeting Antibacterial and Antifouling Properties.

The demand for bio-based and safer composite materials is increasing due to the growth of the industry, human population, and environmental concerns. In this framework, sustainable and safer cork-polymer composites (CPC), based on green low-density polyethylene (LDPE) were developed using melt-based technologies. Chitosan and polyethylene-graft-maleic anhydride (PE-g-MA) were employed to enhance the CPC's properties. The morphology, wettability, mechanical, thermal, and antibacterial properties of the CPC against Pseudomonas putida (P. putida) and Staphylococcus aureus (S. aureus) were examined. The CPC showed improved stiffness when compared with that of the LDPE matrix, preferably when combined with chitosan and PE-g-MA (5 wt. %), reinforcing the stiffness (58.8%) and the strength (66.7%). Chitosan also increased the composite stiffness and strength, as well as reduced the surface hydrophilicity. The CPCs' antibacterial activity revealed that cork significantly reduces the biofilm on the polymer matrix. The highest biofilm reduction was found with CPC containing cork and 5 wt. % chitosan for both P. putida (54% reduction) and S. aureus (36% reduction), confirming their potential to extend the lifespan of products for packaging and healthcare, among other applications. This work leads to the understanding of the factors that influence biofilm formation in cork composites and provides a strategy to reinforce their behavior using chitosan.

Characterization and biofouling potential analysis of two cyanobacterial strains isolated from Cape Verde and Morocco.

Cyanobacteria are new sources of value-added compounds but also ubiquitous and harmful microfoulers on marine biofouling. In this work, the isolation and identification of two cyanobacterial strains isolated from Cape Verde and Morocco, as well as their biofilm-forming ability on glass and Perspex under controlled hydrodynamic conditions, were performed. Phylogenetic analysis revealed that cyanobacterial strains isolated belong to Leptothoe and Jaaginema genera (Leptothoe sp. LEGE 181153 and Jaaginema sp. LEGE 191154). From quantitative and qualitative data of wet weight, chlorophyll a content and biofilm thickness obtained by optical coherence tomography, no significant differences were found in biofilms developed by the same cyanobacterial strain on different surfaces (glass and Perspex). However, the biofilm-forming potential of Leptothoe sp. LEGE 181153 proved to be higher compared with Jaaginema sp. LEGE 191154, particularly at the maturation stage of biofilm development. Three-dimensional biofilm images obtained from confocal laser scanning microscopy showed different patterns between both cyanobacterial strains and also among the two surfaces. Because standard methodologies to evaluate cyanobacterial biofilm formation, as well as two different optical imaging techniques, were used, this work also highlights the possibility of integrating different techniques to evaluate a complex phenomenon like cyanobacterial biofilm development.

Advances on Bacterial and Fungal Biofilms for the Production of Added-Value Compounds.

In recent years, abundant research has been performed on biofilms for the production of compounds with biotechnological and industrial relevance. The use of biofilm platforms has been seen as a compelling approach to producing fine and bulk chemicals such as organic acids, alcohols, and solvents. However, the production of recombinant proteins using this system is still scarce. Biofilm reactors are known to have higher biomass density, operational stability, and potential for long-term operation than suspended cell reactors. In addition, there is an increasing demand to harness industrial and agricultural wastes and biorefinery residues to improve process sustainability and reduce production costs. The synthesis of recombinant proteins and other high-value compounds is mainly achieved using suspended cultures of bacteria, yeasts, and fungi. This review discusses the use of biofilm reactors for the production of recombinant proteins and other added-value compounds using bacteria and fungi.

Antifouling Performance of Carbon-Based Coatings for Marine Applications: A Systematic Review.

Although carbon materials are widely used in surface engineering, particularly graphene (GP) and carbon nanotubes (CNTs), the application of these nanocomposites for the development of antibiofilm marine surfaces is still poorly documented. The aim of this study was, thus, to gather and discuss the relevant literature concerning the antifouling performance of carbon-based coatings against marine micro- and macrofoulers. For this purpose, a PRISMA-oriented systematic review was conducted based on predefined criteria, which resulted in the selection of thirty studies for a qualitative synthesis. In addition, the retrieved publications were subjected to a quality assessment process based on an adapted Methodological Index for Non-Randomized Studies (MINORS) scale. In general, this review demonstrated the promising antifouling performance of these carbon nanomaterials in marine environments. Further, results from the revised studies suggested that functionalized GP- and CNTs-based marine coatings exhibited improved antifouling performance compared to these materials in pristine forms. Thanks to their high self-cleaning and enhanced antimicrobial properties, as well as durability, these functionalized composites showed outstanding results in protecting submerged surfaces from the settlement of fouling organisms in marine settings. Overall, these findings can pave the way for the development of new carbon-engineered surfaces capable of preventing marine biofouling.

Using Lactobacilli to Fight Escherichia coli and Staphylococcus aureus Biofilms on Urinary Tract Devices.

The low efficacy of conventional treatments and the interest in finding natural-based approaches to counteract biofilm development on urinary tract devices have promoted the research on probiotics. This work evaluated the ability of two probiotic strains, Lactobacillus plantarum and Lactobacillus rhamnosus, in displacing pre-formed biofilms of Escherichia coli and Staphylococcus aureus from medical-grade silicone. Single-species biofilms of 24 h were placed in contact with each probiotic suspension for 6 h and 24 h, and the reductions in biofilm cell culturability and total biomass were monitored by counting colony-forming units and crystal violet assay, respectively. Both probiotics significantly reduced the culturability of E. coli and S. aureus biofilms, mainly after 24 h of exposure, with reduction percentages of 70% and 77% for L. plantarum and 76% and 63% for L. rhamnosus, respectively. Additionally, the amount of E. coli biofilm determined by CV staining was maintained approximately constant after 6 h of probiotic contact and significantly reduced up to 67% after 24 h. For S. aureus, only L. rhamnosus caused a significant effect on biofilm amount after 6 h of treatment. Hence, this study demonstrated the potential of lactobacilli to control the development of pre-established uropathogenic biofilms.

A Selection of Platforms to Evaluate Surface Adhesion and Biofilm Formation in Controlled Hydrodynamic Conditions.

The early colonization of surfaces and subsequent biofilm development have severe impacts in environmental, industrial, and biomedical settings since they entail high costs and health risks. To develop more effective biofilm control strategies, there is a need to obtain laboratory biofilms that resemble those found in natural or man-made settings. Since microbial adhesion and biofilm formation are strongly affected by hydrodynamics, the knowledge of flow characteristics in different marine, food processing, and medical device locations is essential. Once the hydrodynamic conditions are known, platforms for cell adhesion and biofilm formation should be selected and operated, in order to obtain reproducible biofilms that mimic those found in target scenarios. This review focuses on the most widely used platforms that enable the study of initial microbial adhesion and biofilm formation under controlled hydrodynamic conditions-modified Robbins devices, flow chambers, rotating biofilm devices, microplates, and microfluidic devices-and where numerical simulations have been used to define relevant flow characteristics, namely the shear stress and shear rate.

Effect of Lactobacillus plantarum Biofilms on the Adhesion of Escherichia coli to Urinary Tract Devices.

Novel technologies to prevent biofilm formation on urinary tract devices (UTDs) are continually being developed, with the ultimate purpose of reducing the incidence of urinary infections. Probiotics have been described as having the ability to displace adhering uropathogens and inhibit microbial adhesion to UTD materials. This work aimed to evaluate the effect of pre-established Lactobacillus plantarum biofilms on the adhesion of Escherichia coli to medical-grade silicone. The optimal growth conditions of lactobacilli biofilms on silicone were first assessed in 12-well plates. Then, biofilms of L. plantarum were placed in contact with E. coli suspensions for up to 24 h under quasi-static conditions. Biofilm monitoring was performed by determining the number of culturable cells and by confocal laser scanning microscopy (CLSM). Results showed significant reductions of 76%, 77% and 99% in E. coli culturability after exposure to L. plantarum biofilms for 3, 6 and 12 h, respectively, corroborating the CLSM analysis. The interactions between microbial cell surfaces and the silicone surface with and without L. plantarum biofilms were also characterized using contact angle measurements, where E. coli was shown to be thermodynamically less prone to adhere to L. plantarum biofilms than to silicone. Thus, this study suggests the use of probiotic cells as potential antibiofilm agents for urinary tract applications.

Assessment of the environmental compatibility and antifouling performance of an innovative biocidal and foul-release multifunctional marine coating.

The control of marine biofouling has raised serious environmental concerns, thus the continuous release of toxic and persistent biocidal agents applied as anti-biofouling coatings have triggered the search for non-toxic strategies. However, most of them still lack rigorous evaluation of their ecotoxicity and antifouling effects under real scenarios and their correlation with simulated assays. In this work, the biocide releasing risk and ecotoxicity of a biocidal and foul-release polydimethylsiloxane (PDMS)-based marine coating containing grafted Econea biocide (<0.6 wt.%) were evaluated under simulated real mechanical wear conditions at a pilot-scale system, and under extreme wear scenarios (washability settings). The coating system demonstrated low environmental impact against the model Vibrio fischeri bacterium and marine algae, associated with the effective biocide grafting in the coating matrix and subsequent biocide release minimization. This multifunctional coating system also showed auspicious antifouling (AF) effects, with an AF performance index significantly higher (API > 89) than a single foul-release system (AF < 40) after two and half years at a real immersion scenario in the Portuguese shore of the Atlantic Ocean. These field results corroborated the antibiofilm performance evaluated with Pseudoalteromonas tunicata at simulated dynamic marine conditions after seven-week assays. This eco-friendly multifunctional strategy, validated by both simulated testing conditions and real field tests, is believed to be a powerful tool for the development of AF technologies and a potential contribution to the quest for new environmentally friendly antifouling solutions.

The Use of Probiotics to Fight Biofilms in Medical Devices: A Systematic Review and Meta-Analysis.

Medical device-associated infections (MDAI) are a critical problem due to the increasing usage of medical devices in the aging population. The inhibition of biofilm formation through the use of probiotics has received attention from the medical field in the last years. However, this sparse knowledge has not been properly reviewed, so that successful strategies for biofilm management can be developed. This study aims to summarize the relevant literature about the effect of probiotics and their metabolites on biofilm formation in medical devices using a PRISMA-oriented (Preferred Reporting Items for Systematic reviews and Meta-Analyses) systematic search and meta-analysis. This approach revealed that the use of probiotics and their products is a promising strategy to hinder biofilm growth by a broad spectrum of pathogenic microorganisms. The meta-analysis showed a pooled effect estimate for the proportion of biofilm reduction of 70% for biosurfactants, 76% for cell-free supernatants (CFS), 77% for probiotic cells and 88% for exopolysaccharides (EPS). This review also highlights the need to properly analyze and report data, as well as the importance of standardizing the in vitro culture conditions to facilitate the comparison between studies. This is essential to increase the predictive value of the studies and translate their findings into clinical applications.

Characterization of planktonic and biofilm cells from two filamentous cyanobacteria using a shotgun proteomic approach.

Cyanobacteria promote marine biofouling with significant impacts. A qualitative proteomic analysis, by LC-MS/MS, of planktonic and biofilm cells from two cyanobacteria was performed. Biofilms were formed on glass and perspex at two relevant hydrodynamic conditions for marine environments (average shear rates of 4 s-1 and 40 s-1). For both strains and surfaces, biofilm development was higher at 4 s-1. Biofilm development of Nodosilinea sp. LEGE 06145 was substantially higher than Nodosilinea sp. LEGE 06119, but no significant differences were found between surfaces. Overall, 377 and 301 different proteins were identified for Nodosilinea sp. LEGE 06145 and Nodosilinea sp. LEGE 06119. Differences in protein composition were more noticeable in biofilms formed under different hydrodynamic conditions than in those formed on different surfaces. Ribosomal and photosynthetic proteins were identified in most conditions. The characterization performed gives new insights into how shear rate and surface affect the planktonic to biofilm transition, from a structural and proteomics perspective.

Biofilm formation behaviour of marine filamentous cyanobacterial strains in controlled hydrodynamic conditions.

Marine biofouling has severe economic impacts and cyanobacteria play a significant role as early surface colonizers. Despite this fact, cyanobacterial biofilm formation studies in controlled hydrodynamic conditions are scarce. In this work, computational fluid dynamics was used to determine the shear rate field on coupons that were placed inside the wells of agitated 12-well microtiter plates. Biofilm formation by three different cyanobacterial strains was assessed at two different shear rates (4 and 40 s-1 ) which can be found in natural ecosystems and using different surfaces (glass and perspex). Biofilm formation was higher under low shear conditions, and differences obtained between surfaces were not always statistically significant. The hydrodynamic effect was more noticeable during the biofilm maturation phase rather than during initial cell adhesion and optical coherence tomography showed that different shear rates can affect biofilm architecture. This study is particularly relevant given the cosmopolitan distribution of these cyanobacterial strains and the biofouling potential of these organisms.

Evaluating Efficacy of Antimicrobial and Antifouling Materials for Urinary Tract Medical Devices: Challenges and Recommendations.

In Europe, the mean incidence of urinary tract infections in intensive care units is 1.1 per 1000 patient-days. Of these cases, catheter-associated urinary tract infections (CAUTI) account for 98%. In total, CAUTI in hospitals is estimated to give additional health-care costs of £1-2.5 billion in the United Kingdom alone. This is in sharp contrast to the low cost of urinary catheters and emphasizes the need for innovative products that reduce the incidence rate of CAUTI. Ureteral stents and other urinary-tract devices suffer similar problems. Antimicrobial strategies are being developed, however, the evaluation of their efficacy is very challenging. This review aims to provide considerations and recommendations covering all relevant aspects of antimicrobial material testing, including surface characterization, biocompatibility, cytotoxicity, in vitro and in vivo tests, microbial strain selection, and hydrodynamic conditions, all in the perspective of complying to the complex pathology of device-associated urinary tract infection. The recommendations should be on the basis of standard assays to be developed which would enable comparisons of results obtained in different research labs both in industry and in academia, as well as provide industry and academia with tools to assess the antimicrobial properties for urinary tract devices in a reliable way.

Time-course determination of plasmid content in eukaryotic and prokaryotic cells using real-time PCR.

A Real-Time PCR method was developed to monitor the plasmid copy number (PCN) in Escherichia coli and Chinese hamster ovary (CHO) cells. E. coli was transformed with plasmids containing a ColE1 or p15A origin of replication and CHO cells were transfected with a ColE1 derived plasmid used in DNA vaccination and carrying the green fluorescent protein (GFP) reporter gene. The procedure requires neither specific cell lysis nor DNA purification and can be performed in <30 min with dynamic ranges covering 0.9 pg-55 ng, and 5.0 pg-2.5 ng of plasmid DNA (pDNA) for E. coli and CHO cells, respectively. Analysis of PCN in E. coli batch cultures revealed that the maximum copy number per cell is attained in mid-exponential phase and that this number decreases on average 80% towards the end of cultivation for both types of plasmids. The plasmid content of CHO cells determined 24 h post-transfection was around 3 x 104 copies per cell although only 37% of the cells expressed GFP one day after transfection. The half-life of pDNA was 20 h and around 100 copies/cell were still detected 6 days after transfection.

Evaluation of inducible promoters on the secretion of a ZZ-proinsulin fusion protein in Escherichia coli.

Four inducible promoters, uspA, uspB, lacUV5 and malK, were evaluated in the expression of the fusion protein ZZ-proinsulin by Escherichia coli. The aim was to select for their effects on the most appropriate expression system (promoter and culture medium) for secretion of ZZ-proinsulin to the periplasmic space and culture medium. All the expression vectors contained the RNase III cleavage site to ensure that the mRNA translation rate remained independent of 5'-untranslated regions thus making promoter strength comparisons more accurate. The highest ZZ-proinsulin secretion yields were 6.2 mg/g of dry cell weight in the periplasmic space and 2.6 mg/g of dry cell weight in the culture medium using the malK promoter. It was also demonstrated that the use of M9 minimal medium favours secretion.