Electrochemical Aptasensor for the Detection of the Key Virulence Factor YadA of Yersinia enterocolitica.

New point-of-care (POC) diagnosis of bacterial infections are imperative to overcome the deficiencies of conventional methods, such as culture and molecular methods. In this study, we identified new aptamers that bind to the virulence factor Yersinia adhesin A (YadA) of Yersinia enterocolitica using cell-systematic evolution of ligands by exponential enrichment (cell-SELEX). Escherichia coli expressing YadA on the cell surface was used as a target cell. After eight cycles of selection, the final aptamer pool was sequenced by high throughput sequencing using the Illumina Novaseq platform. The sequencing data, analyzed using the Geneious software, was aligned, filtered and demultiplexed to obtain the key nucleotides possibly involved in the target binding. The most promising aptamer candidate, Apt1, bound specifically to YadA with a dissociation constant (Kd) of 11 nM. Apt1 was used to develop a simple electrochemical biosensor with a two-step, label-free design towards the detection of YadA. The sensor surface modifications and its ability to bind successfully and stably to YadA were confirmed by cyclic voltammetry, impedance spectroscopy and square wave voltammetry. The biosensor enabled the detection of YadA in a linear range between 7.0 × 104 and 7.0 × 107 CFU mL−1 and showed a square correlation coefficient >0.99. The standard deviation and the limit of detection was ~2.5% and 7.0 × 104 CFU mL−1, respectively. Overall, the results suggest that this novel biosensor incorporating Apt1 can potentially be used as a sensitive POC detection system to aid the diagnosis of Y. enterocolitica infections. Furthermore, this simple yet innovative approach could be replicated to select aptamers for other (bacterial) targets and to develop the corresponding biosensors for their detection.

Novel Biorecognition Elements against Pathogens in the Design of State-of-the-Art Diagnostics.

Infectious agents, especially bacteria and viruses, account for a vast number of hospitalisations and mortality worldwide. Providing effective and timely diagnostics for the multiplicity of infectious diseases is challenging. Conventional diagnostic solutions, although technologically advanced, are highly complex and often inaccessible in resource-limited settings. An alternative strategy involves convenient rapid diagnostics which can be easily administered at the point-of-care (POC) and at low cost without sacrificing reliability. Biosensors and other rapid POC diagnostic tools which require biorecognition elements to precisely identify the causative pathogen are being developed. The effectiveness of these devices is highly dependent on their biorecognition capabilities. Naturally occurring biorecognition elements include antibodies, bacteriophages and enzymes. Recently, modified molecules such as DNAzymes, peptide nucleic acids and molecules which suffer a selective screening like aptamers and peptides are gaining interest for their biorecognition capabilities and other advantages over purely natural ones, such as robustness and lower production costs. Antimicrobials with a broad-spectrum activity against pathogens, such as antibiotics, are also used in dual diagnostic and therapeutic strategies. Other successful pathogen identification strategies use chemical ligands, molecularly imprinted polymers and Clustered Regularly Interspaced Short Palindromic Repeats-associated nuclease. Herein, the latest developments regarding biorecognition elements and strategies to use them in the design of new biosensors for pathogens detection are reviewed.

An Update on Wastewater Multi-Resistant Bacteria: Identification of Clinical Pathogens Such as Escherichia coli O25b:H4-B2-ST131-Producing CTX-M-15 ESBL and KPC-3 Carbapenemase-Producing Klebsiella oxytoca.

Wastewater treatment plants (WWTPs) are significant reservoirs of bacterial resistance. This work aims to identify the determinants of resistance produced by Gram-negative bacteria in the influent and effluent of two WWTPs in Portugal. A total of 96 wastewater samples were obtained between 2016 and 2019. The numbers of total aerobic and fecal contamination bacteria were evaluated, and genomic features were searched by polymerase chain reaction (PCR) and Next-Generation Sequencing (NGS). Enterobacteriaceae corresponded to 78.6% (n = 161) of the 205 isolates identified by 16sRNA. The most frequent isolates were Escherichia spp. (57.1%, n = 117), followed by Aeromonas spp. (16.1%, n = 33) and Klebsiella spp. (12.7%, n = 26). The remaining 29 isolates (14.1%) were distributed across 10 different genera. Among the 183 resistant genes detected, 54 isolates produced extended spectrum ß-lactamases (ESBL), of which blaCTX-M-15 was predominant (37 isolates; 68.5%). A KPC-3 carbapenemase-producing K. oxytoca was identified (n = 1), with blaKPC-3 included in a transposon Tn4401 isoform b. A higher number of virulence genes (VG) (19 genes) was found in the E. coli 5301 (O25b-ST131-B2) isolate compared with a commensal E. coli 5281 (O25b-ST410-A) (six genes). Both shared five VG [Enterobactin; Aerobactin, CFA/1 (clade α); Type1 (clade γ1); Type IV]. In conclusion, this work highlights the role of relevant clinical bacteria in WWTPs, such as KPC-3-producing K. oxytoca, and, for the first time, a CTX-M-15-producing Ochromobactrum intermedium, a human opportunistic pathogen, and a SED-1-producing Citrobacter farmeri, an uncommon CTX-M-type extended-spectrum beta-lactamase.

Peptide structure: Its effect on penetration into human hair.

The influence of the peptide structure on its penetration inside hair was studied, together with the effect of hair bleaching (oxidation). For that reason, the outcome of positioning a charged sequence (KAKAK) either at the N or C terminal on hair penetration has been studied for peptides with 17 residues each. It was observed that the penetration of these peptides into hair was driven by electrostatic interactions, where the position of the charged group at the peptide structure was of major importance. The penetration was only achieved for damaged hair due to its higher negative charge at the membrane surface. It was also observed that the peptides were able to restore the original tensile strength of bleached hair. Consequently, the knowledge of hair surface properties is of extreme importance when designing peptides directed for hair treatment.

Antibacterial Electrospun Poly(vinyl alcohol)/Enzymatic Synthesized Poly(catechol) Nanofibrous Midlayer Membrane for Ultrafiltration.

Two different nanofibrous antibacterial membranes containing enzymatically synthesized poly(catechol) (PC) or silver nitrate (AgNO3, positive control) blended with poly(vinyl alcohol) (PVA) and electrospun onto a poly(vinylidene fluoride) (PVDF) basal disc to generate thin-film composite midlayers were produced for water ultrafiltration applications. The developed membranes were thoroughly characterized in terms of morphology, chemical composition, and general mechanical and thermal features, antimicrobial activity, and ultrafiltration capabilities. The electrospun blends were recognized as homogeneous. Data revealed relevant conformational changes in the PVA side groups, attributed to hydrogen bonding, high thermal stability, and residual mass. PVDF+PVA/AgNO3 membrane displayed 100% growth inhibition of both Gram-positive and Gram-negative bacteria strains, despite the wide range of fiber diameters generated, from 24 to 125 nm, formation of numerous beads, and irregular morphology. The PVDF+PVA/PC membrane showed a good growth inhibition of Staphylococcus aureus (92%) and revealed a smooth morphology with no relevant bead formations and diameters ranging from 68 to 131 nm. The ultrafiltration abilities of the membrane containing PVA/PC were tested in a dead-end high-pressure cell (4 bar) using a reactive dye in distilled water and seawater. After 5 cycles, a maximum rejection of ≈85% with an average flux rate of 70 L m-2 h-1 for distilled water and ≈64% with an average flux rate of 62 L m-2 h-1 for seawater were determined with an overall salt rejection of ≈5%.

Silk-based anisotropical 3D biotextiles for bone regeneration.

Bone loss in the craniofacial complex can been treated using several conventional therapeutic strategies that face many obstacles and limitations. In this work, novel three-dimensional (3D) biotextile architectures were developed as a possible strategy for flat bone regeneration applications. As a fully automated processing route, this strategy as potential to be easily industrialized. Silk fibroin (SF) yarns were processed into weft-knitted fabrics spaced by a monofilament of polyethylene terephthalate (PET). A comparative study with a similar 3D structure made entirely of PET was established. Highly porous scaffolds with homogeneous pore distribution were observed using micro-computed tomography analysis. The wet state dynamic mechanical analysis revealed a storage modulus In the frequency range tested, the storage modulus values obtained for SF-PET scaffolds were higher than for the PET scaffolds. Human adipose-derived stem cells (hASCs) cultured on the SF-PET spacer structures showed the typical pattern for ALP activity under osteogenic culture conditions. Osteogenic differentiation of hASCs on SF-PET and PET constructs was also observed by extracellular matrix mineralization and expression of osteogenic-related markers (osteocalcin, osteopontin and collagen type I) after 28 days of osteogenic culture, in comparison to the control basal medium. The quantification of convergent macroscopic blood vessels toward the scaffolds by a chick chorioallantoic membrane assay, showed higher angiogenic response induced by the SF-PET textile scaffolds than PET structures and gelatin sponge controls. Subcutaneous implantation in CD-1 mice revealed tissue ingrowth's accompanied by blood vessels infiltration in both spacer constructs. The structural adaptability of textile structures combined to the structural similarities of the 3D knitted spacer fabrics to craniofacial bone tissue and achieved biological performance, make these scaffolds a possible solution for tissue engineering approaches in this area.

Modulating cell adhesion to polybutylene succinate biotextile constructs for tissue engineering applications.

Textile-based technologies are powerful routes for the production of three-dimensional porous architectures for tissue engineering applications because of their feasibility and possibility for scaling-up. Herein, the use of knitting technology to produce polybutylene succinate fibre-based porous architectures is described. Furthermore, different treatments have been applied to functionalize the surface of the scaffolds developed: sodium hydroxide etching, ultraviolet radiation exposure in an ozone atmosphere and grafting (acrylic acid, vinyl phosphonic acid and vinyl sulphonic acid) after oxygen plasma activation as a way to tailor cell adhesion. A possible effect of the applied treatments on the bulk properties of the textile scaffolds has been considered and thus tensile tests in dry and hydrated states were also carried out. The microscopy results indicated that the surface morphology and roughness were affected by the applied treatments. The X-ray photoelectron spectroscopy and contact angle measurements showed the incorporation of oxygen-containing groups and higher surface free energy as result of the surface treatments applied. The DNA quantification and scanning electron microscopy analysis revealed that these modifications enhanced cell adhesion and altered cell morphology. Generally, sodium hydroxide treatment altered most significantly the surface properties, which in turn resulted in a high number of cells adherent to these surfaces. Based on the results obtained, the proposed surface treatments are appropriate to modify polybutylene succinate knitting scaffolds, influencing cell adhesion and its potential for use in tissue engineering applications. Copyright © 2016 John Wiley & Sons, Ltd.

Laccase immobilization on bacterial nanocellulose membranes: Antimicrobial, kinetic and stability properties.

This work studied the physical immobilization of a commercial laccase on bacterial nanocellulose (BNC) aiming to identify the laccase antibacterial properties suitable for wound dressings. Physico-chemical analysis demonstrates that the BNC structure is manly formed by pure crystalline Iα cellulose. The pH optimum and activation energy of free laccase depends on the substrate employed corresponding to pH 6, 7, 3 and 57, 22, 48kJmol(-1) for 2,6-dimethylphenol (DMP), catechol and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), respectively. The Michaelis-Menten constant (Km) value for the immobilized laccase (0.77mM) was found to be almost double of that of the free enzyme (0.42mM). However, the specific activities of immobilized and free laccase are similar suggesting that the cage-like structure of BNC allows entrapped laccase to maintain some flexibility and favour substrate accessibility. The results clearly show the antimicrobial effect of laccase in Gram-positive (92%) and Gram-negative (26%) bacteria and cytotoxicity acceptable for wound dressing applications.

Influence of different surface modification treatments on silk biotextiles for tissue engineering applications.

Biotextile structures from silk fibroin have demonstrated to be particularly interesting for tissue engineering (TE) applications due to their high mechanical strength, interconnectivity, porosity, and ability to degrade under physiological conditions. In this work, we described several surface treatments of knitted silk fibroin (SF) scaffolds, namely sodium hydroxide (NaOH) solution, ultraviolet radiation exposure in an ozone atmosphere (UV/O3) and oxygen (O2) plasma treatment followed by acrylic acid (AAc), vinyl phosphonic acid (VPA), and vinyl sulfonic acid (VSA) immersion. The effect of these treatments on the mechanical properties of the textile constructs was evaluated by tensile tests in dry and hydrated states. Surface properties such as morphology, topography, wettability and elemental composition were also affected by the applied treatments. The in vitro biological behavior of L929 fibroblasts revealed that cells were able to adhere and spread both on the untreated and surface-modified textile constructs. The applied treatments had different effects on the scaffolds' surface properties, confirming that these modifications can be considered as useful techniques to modulate the surface of biomaterials according to the targeted application.

Screen-Printed Photochromic Textiles through New Inks Based on SiO2@naphthopyran Nanoparticles.

Photochromic silica nanoparticles (SiO2@NPT), fabricated through the covalent immobilization of silylated naphthopyrans (NPTs) based on 2H-naphtho[1,2-b]pyran (S1, S2) and 3H-naphtho[2,1-b]pyran (S3, S4) or through the direct adsorption of the parent naphthopyrans (1, 3) onto silica nanoparticles (SiO2 NPs), were successfully incorporated onto cotton fabrics by a screen-printing process. Two aqueous acrylic- (AC-) and polyurethane- (PU-) based inks were used as dispersing media. All textiles exhibited reversible photochromism under UV and solar irradiation, developing fast responses and intense coloration. The fabrics coated with SiO2@S1 and SiO2@S2 showed rapid color changes and high contrasts (ΔE*ab = 39-52), despite presenting slower bleaching kinetics (2-3 h to fade to the original color), whereas the textiles coated with SiO2@S3 and SiO2@S4 exhibited excellent engagement between coloration and decoloration rates (coloration and fading times of 1 and 2 min, respectively; ΔE*ab = 27-53). The PU-based fabrics showed excellent results during the washing fastness tests, whereas the AC-based textiles evidenced good results only when a protective transfer film was applied over the printed design.

Preparation and characterization of polysaccharides/PVA blend nanofibrous membranes by electrospinning method.

A series of polyvinyl alcohol (PVA), PVA/chitosan (CS) and PVA/cyanobacterial extracellular polymeric substances (EPS) blended nanofibrous membranes were produced by electrospinning using a microfiltration poly(vinylidene fluoride) (PVDF) basal membrane, for potential applications in water filtration. Nanofibres were obtained from solutions of 20% (w/w) PVA with 1% (w/w) CS or EPS, using a weight ratio of 60/40. Blended nanofibres have shown a smooth morphology, no beads formation and diameters between 50 and 130 nm. Thermo-mechanical analysis demonstrated that there were inter and/or intramolecular hydrogen bonds between the molecules of PVA/CS and PVA/EPS in the blends. The electrospun blended PVA/EPS membrane showed better tensile mechanical properties when compared with PVA and PVA/CS, and resisted more against disintegration in the temperature range between 10 and 50 °C. Finally, the blended membranes have shown an increase in chromium binding capacity of 5%. This is the first successful report of a blended membrane of electrospinned cyanobacterial polysaccharide with PVA.

Production and characterization of extracellular carbohydrate polymer from Cyanothece sp. CCY 0110.

Cyanobacterial extracellular polymeric substances (EPS) are heteropolysaccharides that possess characteristics suitable for industrial applications, notably a high number of different monomers, strong anionic nature and high hydrophobicity. However, systematic studies that unveil the conditions influencing EPS synthesis and/or its characteristics are mandatory. In this work, Cyanothece sp. CCY 0110 was used as model organism. Our results revealed that this strain is among the most efficient EPS producers, and that the amount of RPS (released polysaccharides) is mainly related to the number of cells, rather than to the amount produced by each cell. Light was the key parameter, with high light intensity enhancing significantly RPS production (reaching 1.8 g L(-1)), especially in the presence of combined nitrogen. The data showed that RPS are composed by nine different monosaccharides (including two uronic acids), the presence of sulfate groups and peptides, and that the polymer is remarkably thermostable and amorphous in nature.

New biotextiles for tissue engineering: development, characterization and in vitro cellular viability.

This work proposes biodegradable textile-based structures for tissue engineering applications. We describe the use of two polymers, polybutylene succinate (PBS) proposed as a viable multifilamentand silk fibroin (SF), to produce fibre-based finely tuned porous architectures by weft knitting. PBS is here proposed as a viable extruded multifilament fibre to be processed by a textile-based technology. A comparative study was undertaken using a SF fibre with a similar linear density. The knitted constructs obtained are described in terms of their morphology, mechanical properties, swelling capability, degradation behaviour and cytotoxicity. The weft knitting technology used offers superior control over the scaffold design (e.g. size, shape, porosity and fibre alignment), manufacturing and reproducibility. The presented fibres allow the processing of a very reproducible intra-architectural scaffold geometry which is fully interconnected, thus providing a high surface area for cell attachment and tissue in-growth. The two types of polymer fibre allow the generation of constructs with distinct characteristics in terms of the surface physico-chemistry, mechanical performance and degradation capability, which has an impact on the resulting cell behaviour at the surface of the respective biotextiles. Preliminary cytotoxicity screening showed that both materials can support cell adhesion and proliferation. These results constitute a first validation of the two biotextiles as viable matrices for tissue engineering prior to the development of more complex systems. Given the processing efficacy and versatility of the knitting technology and the interesting structural and surface properties of the proposed polymer fibres it is foreseen that the developed systems could be attractive for the functional engineering of tissues such as skin, ligament, bone or cartilage.

Fermentation performance of Candida guilliermondii for xylitol production on single and mixed substrate media.

Semidefined media fermentation simulating the sugar composition of hemicellulosic hydrolysates (around 85 g l(-1) xylose, 17 g l(-1) glucose, and 9 g l(-1) arabinose) was investigated to evaluate the glucose and arabinose influence on xylose-to-xylitol bioconversion by Candida guilliermondii. The results revealed that glucose reduced the xylose consumption rate by 30%. Arabinose did not affect the xylose consumption but its utilization by the yeast was fully repressed by both glucose and xylose sugars. Arabinose was only consumed when it was used as a single carbon source. Xylitol production was best when glucose was not present in the fermentation medium. On the other hand, the arabinose favored the xylitol yield (which attained 0.74 g g(-1) xylose consumed) and it did not interfere with xylitol volumetric productivity (Q(P) = 0.85 g g(-1)), the value of which was similar to that obtained with xylose alone.

Detergent formulations for wool domestic washings containing immobilized enzymes.

The stability of immobilized and native Esperase, a commercial serine protease, was studied by incubating the enzymes in four formulations containing the same amount of anionic and non-ionic surfactants. The results show that the activity of the immobilized enzyme is not affected by the presence of detergents while the native enzyme lost 50% of activity after 20 min of incubation in these four formulations. The washing performance of the detergents prepared with the immobilized Esperase was studied on cotton and wool fabric samples stained with human blood and egg yolk, using as control the detergent containing native Esperase. The best stain removal for cotton samples stained with human blood was achieved using the detergent with immobilized Esperase. Several physical tests confirmed that wool keratin was not degraded by the immobilized Esperase, validating the ability to use formulated detergents containing this immobilized enzyme for safe wool domestic washing.