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Hydrogels based on natural polysaccharides can have unique properties and be tailored for several applications, which may be mainly limited by the fragile structure and weak mechanical properties of this type of system. We successfully prepared cryogels made of newly synthesized kefiran exopolysaccharide-chondroitin sulfate (CS) conjugate via carbodiimide-mediated coupling to overcome these drawbacks. The freeze-thawing procedure of cryogel preparation followed by lyophilization is a promising route to fabricate polymer-based scaffolds with countless and valuable biomedical applications. The novel graft macromolecular compound (kefiran-CS conjugate) was characterized through 1 H-NMR and FTIR spectroscopy-which confirmed the structure of the conjugate, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA)-which mirrored good thermal stability (degradation temperature of about 215 °C) and, finally, gel permeation chromatography-size exclusion chromatography (GPC-SEC)-which proved an increased molecular weight due to chemical coupling of kefiran with CS. At the same time, the corresponding cryogels physically crosslinked after the freeze-thawing procedure were investigated by scanning electron microscopy (SEM), Micro-CT, and dynamic rheology. The results revealed a prevalent contribution of elastic/storage component to the viscoelastic behavior of cryogels in swollen state, a micromorphology with micrometer-sized open pores fully interconnected, and high porosity (ca. 90%) observed for freeze-dried cryogels. Furthermore, the metabolic activity and proliferation of human adipose stem cells (hASCs), when cultured onto the developed kefiran-CS cryogel, was maintained at a satisfactory level over 72 h. Based on the results obtained, it can be inferred that the newly freeze-dried kefiran-CS cryogels possess a host of unique properties that render them highly suitable for use in tissue engineering, regenerative medicine, drug delivery, and other biomedical applications where robust mechanical properties and biocompatibility are crucial.

Kefiran is an exopolysaccharide produced by the microflora of kefir grains used to produce the fermented milk beverage kefir. The health-promoting and physicochemical properties of kefiran led to its exploration for a range of applications, mainly in the food industry and biomedical fields. Aiming to explore its potential for tissue engineering and regenerative medicine (TERM) applications, the kefiran biopolymer obtained through three different extraction methodologies was fully characterized and compared. High-quality kefiran polysaccharides were recovered with suitable yield through different extraction protocols. The methods consisted of heating the kefir grains prior to recovering kefiran by centrifugation and differed mainly in the precipitation steps included before lyophilization. Then, kefiran scaffolds were successfully produced from each extract by cryogelation and freeze-drying. In all extracts, it was possible to identify the molecular structure of the kefiran polysaccharide through 1 H-NMR and FTIR spectra. The kefiran from extraction 1 showed the highest molecular weight (~3000 kDa) and the best rheological properties, showing a pseudoplastic behavior; its scaffold presented the highest value of porosity (93.2% ± 2), and wall thickness (85.8 µm ± 16.3). All extracts showed thermal stability, good injectability and desirable viscoelastic properties; the developed scaffolds demonstrated mechanical stability, elastic behavior, and pore size comprised between 98-94 µm. Additionally, all kefiran products proved to be non-cytotoxic over L929 cells. The interesting structural, physicochemical, and biological properties showed by the kefiran extracts and cryogels revealed their biomedical potential and suitability for TERM applications.

Hydrogel application feasibility is still limited mainly due to their low mechanical strength and fragile nature. Therefore, several physical and chemical cross-linking modifications are being used to improve their properties. In this research, methacrylated Kefiran was synthesized by reacting Kefiran with methacrylic anhydride (MA). The developed MA-Kefiran was physicochemically characterized, and its biological properties evaluated by different techniques. Chemical modification of MA-Kefiran was confirmed by 1 H-NMR and FTIR and GPC-SEC showed an average Mw of 793 kDa (PDI 1.3). The mechanical data obtained revealed MA-Kefiran to be a pseudoplastic fluid with an extrusion force of 11.21 ± 2.87 N. Moreover, MA-Kefiran 3D cryogels were successfully developed and fully characterized. Through micro-CT and SEM, the scaffolds revealed high porosity (85.53 ± 0.15%) and pore size (33.67 ± 3.13 μm), thick pore walls (11.92 ± 0.44 μm) and a homogeneous structure. Finally, MA-Kefiran revealed to be biocompatible by presenting no hemolytic activity and an improved cellular function of L929 cells observed through the AlamarBlue ® assay. By incorporating methacrylate groups in the Kefiran polysaccharide chain, a MA-Kefiran product was developed with remarkable physical, mechanical, and biological properties, resulting in a promising hydrogel to be used in tissue engineering and regenerative medicine applications.

Kefiran from kefir grains, an exopolysaccharide (EPS) produced by lactic acid bacteria (LAB), has received an increasing interest because of its safe status. This natural biopolymer is a water-soluble glucogalactan with probed health-promoting properties. However, its biological performance has yet to be completely recognized and properly exploited. This research was carried out to evaluate the in vitro antioxidant and the in vitro anti-inflammatory properties of Kefiran biopolymer. Regarding antioxidant activity, the results demonstrated that the Kefiran extract possessed the strongest reducing power and superoxide radical scavenging, over hyaluronic acid (HA, gold standard viscosupplementation treatment). This exopolysaccharide showed a distinct antioxidant performance in the majority of in vitro working mechanisms of antioxidant activity comparing to HA. Moreover, Kefiran presented an interesting capacity to scavenge nitric oxide radical comparing to the gold standard that did not present any potency. Finally, the cytotoxic effects of Kefiran extracts on hASCs were also performed and demonstrated no cytotoxic response, ability to improve cellular function of hASCs. This study demonstrated that Kefiran represented a great scavenger for reactive oxygen and nitrogen species and showed also that it could be an excellent candidate to promote tissue repair and regeneration.

Timely and spatially-regulated injectable hydrogels, able to suppress growing tumors in response to conformational transitions of proteins, are of great interest in cancer research and treatment. Herein, we report rapidly responsive silk fibroin (SF) hydrogels formed by a horseradish peroxidase (HRP) crosslinking reaction at physiological conditions, and demonstrate their use as an artificial biomimetic three-dimensional (3D) matrix. The proposed SF hydrogels presented a viscoelastic nature of injectable hydrogels and spontaneous conformational changes from random coil to β-sheet conformation under physiological conditions. A human neuronal glioblastoma (U251) cell line was used for screening cell encapsulation and in vitro evaluation within the SF hydrogels. The transparent random coil SF hydrogels promoted cell viability and proliferation up to 10 days of culturing, while the crystalline SF hydrogels converted into β-sheet structure induced the formation of TUNEL-positive apoptotic cells. Therefore, this work provides a powerful tool for the investigation of the microenvironment on the programed tumor cells death, by using rapidly responsive SF hydrogels as 3D in vitro tumor models.

Given the significant spatial and temporal heterogeneity in antimicrobial resistance distribution and the factors that affect its evolution, dissemination, and persistence, it is important to highlight that antimicrobial resistance must be viewed as an ecological problem. Monitoring the resistance prevalence of indicator bacteria such as Escherichia coli and enterococci in wild animals makes it possible to show that wildlife has the potential to serve as an environmental reservoir and melting pot of bacterial resistance. These researchers address the issue of antimicrobial-resistant microorganism proliferation in the environment and the related potential human health and environmental impact.

A total of 36 Escherichia coli and 31 enterococci isolates were recovered from 42 common buzzard faecal samples. The E. coli isolates showed high levels of resistance to streptomycin and tetracycline. The following resistance genes were detected: bla(TEM) (20 of 22 ampicillin-resistant isolates), tet(A) and/or tet(B) (16 of 27 tetracycline-resistant isolates), aadA1 (eight of 27 streptomycin-resistant isolates), cmlA (three of 15 chloramphenicol-resistant isolates), aac(3)-II with/without aac(3)-IV (all seven gentamicin-resistant isolates) and sul1 and/or sul2 and/or sul3 [all eight sulfamethoxazole/trimethoprim-resistant (SXT) isolates]. intI1 and intI2 genes were detected in four SXT-resistant isolates. The virulence-associated genes fimA (type 1 fimbriae), papC (P fimbriae) and aer (aerobactin) were detected in 61.1, 13.8 and 11.1% of the isolates, respectively. The isolates belonged to phylogroups A (47.2%), B1 (8.3%), B2 (13.9%) and D (30.5%). For the enterococci isolates, Enterococcus faecium was the most prevalent species (48.4%). High levels of tetracycline and erythromycin resistance were found among our isolates (87 and 81%, respectively). Most of the tetracycline-resistant strains carried the tet(M) and/or tet(L) genes. The erm(B) gene was detected in 80% of erythromycin-resistant isolates. The vat(D) and/or vat(E) genes were found in nine of the 17 quinupristin-dalfopristin-resistant isolates. The enterococcal isolates showing high-level resistance for kanamycin, gentamicin and streptomycin contained the aph(3')-IIIa, aac(6')-aph(2″) and ant(6)-Ia genes, respectively. This report reveals that common buzzards seem to represent an important reservoir, or at least a source, of multi-resistant E. coli and enterococci isolates, and consequently may represent a considerable hazard to human and animal health by transmission of these isolates to waterways and other environmental sources via their faecal deposits.

Vancomycin-resistant Enterococcus faecalis, E. faecium and E. durans isolates with the genotype vanA were detected in 7 of 118 faecal samples (5.9%) of natural gilthead seabream recovered off the coast of Portugal, and one vancomycin-resistant isolate/sample was further characterized. The genes erm(B), tet(L), tet(M), aac(6')-aph(2"), aph(3')-IIIa and/or ant(6)-Ia were identified in most of the 7 vancomycin-resistant enterococci. Sequence types ST273, ST313 and ST76 were detected in three E. faecium isolates and ST6 in two E. faecalis isolates. VanA-containing enterococci are suggested to be disseminated in fish in marine ecosystems close to areas of human activity.

Background: Enterococci have emerged as the third most common cause of nosocomial infections, requiring bactericidal antimicrobial therapy. Although vancomycin resistance is a major problem in clinics and has emerged in an important extend in farm animals, few studies have examined it in wild animals. To determine the prevalence of vanA-containing Enterococcus strains among faecal samples of Seagulls (Larus cachinnans) of Berlengas Natural Reserve of Portugal, we developed a proteomic approach integrated with genomic data. The purpose was to detect the maximum number of proteins that vary in different enterococci species which are thought to be connected in some, as yet unknown, way to antibiotic resistance., Results: From the 57 seagull samples, 54 faecal samples showed the presence of Enterococcus isolates (94.7%). For the enterococci, E. faecium was the most prevalent species in seagulls (50%), followed by E. faecalis and E. durans (10.4%), and E. hirae (6.3%). VanA-containing enterococcal strains were detected in 10.5% of the 57 seagull faecal samples studied. Four of the vanA-containing enterococci were identified as E. faecium and two as E. durans. The tet(M) gene was found in all five tetracycline-resistant vanA strains. The erm(B) gene was demonstrated in all six erythromycin-resistant vanA strains. The hyl virulence gene was detected in all four vanA-containing E. faecium isolates in this study, and two of them harboured the purK1 allele. In addition these strains also showed ampicillin and ciprofoxacin resistance. The whole-cell proteomic profile of vanA-containing Enterococcus strains was applied to evaluate the discriminatory power of this technique for their identification. The major differences among species-specific profiles were found in the positions corresponding to 97-45 kDa. Sixty individualized protein spots for each vanA isolate was identified and suitable for peptide mass fingerprinting measures by spectrometry measuring (MALDI/TOF MS) and their identification through bioinformatic databases query. The proteins were classified in different groups according to their biological function: protein biosynthesis, ATP synthesis, glycolysis, conjugation and antibiotic resistance. Taking into account the origin of these strains and its relation to infectious processes in humans and animals, it is important to explore the proteome of new strains which might serve as protein biomarkers for biological activity., Conclusions: The comprehensive description of proteins isolated from vancomycin-resistant Enterococcus faecium and E. durans may provide new targets for development of antimicrobial agents. This knowledge may help to identify new biomarkers of antibiotic resistance and virulence factors.

Extended-spectrum beta-lactamase-containing Escherichia coli isolates were detected in 32 of 119 fecal samples (26.9%) from birds of prey at Serra da Estrela, and these isolates contained the following beta-lactamases: CTX-M-1 (n = 13), CTX-M-1 plus TEM-1 (n = 14), CTX-M-1 plus TEM-20 (n = 1), SHV-5 (n = 1), SHV-5 plus TEM-1 (n = 2), and TEM-20 (n = 1).

Escherichia coli isolates containing the following extended-spectrum beta-lactamases have been detected in 11 of 57 fecal samples (19.3%) in Berlengas Island seagulls: TEM-52 (eight isolates), CTX-M-1 (one isolate), CTX-M-14a (one isolate), and CTX-M-32 (one isolate). Most of the extended-spectrum beta-lactamase-positive isolates harbored class 1 or class 2 integrons, which included different antibiotic resistance gene cassettes.