Strategies to prevent the occurrence of resistance against antibiotics by using advanced materials

This is a post-peer-review, pre-copyedit version of an article published in Applied microbiology and biotechnology The final authenticated version is available online at: http://dx.doi.org/10.1007/s00253-018-8776-0Drug resistance occurrence is a global healthcare concern responsible for the increased morbidity and mortality in hospitals, time of hospitalisation and huge financial loss. The failure of the most antibiotics to kill Bsuperbugs^ poses the urgent need to develop innovative strategies aimed at not only controlling bacterial infection but also the spread of resistance. The prevention of pathogen host invasion by inhibiting bacterial virulence and biofilm formation, and the utilisation of bactericidal agents with different mode of action than classic antibiotics are the two most promising new alternative strategies to overcome antibiotic resistance. Based on these novel approaches, researchers are developing different advanced materials (nanoparticles, hydrogels and surface coatings) with novel antimicrobial properties. In this review, we summarise the recent advances in terms of engineered materials to prevent bacteria-resistant infections according to the antimicrobial strategies underlying their design.Peer ReviewedPostprint (author's final draft

Antibacterial properties and mechanisms of action of sonoenzymatically synthesized lignin-based nanoparticles

In recent years, lignin has drawn increasing attention for different applications due to its intrinsic antibacterial and antioxidant properties, coupled with biodegradability and biocompatibility. However, chemical modification or combination with metals is usually required to increase its antimicrobial functionality and produce biobased added-value materials for applications wherein bacterial growth should be avoided, such as biomedical and food industries. In this work, a sonoenzymatic approach for the simultaneous functionalization and nanotransformation of lignin to prepare metal-free antibacterial phenolated lignin nanoparticles (PheLigNPs) is developed. The grafting of tannic acid, a natural phenolic compound, onto lignin was achieved by an environmentally friendly approach using laccase oxidation upon the application of high-intensity ultrasound to rearrange lignin into NPs. PheLigNPs presented higher antibacterial activity than nonfunctionalized LigNPs and phenolated lignin in the bulk form, indicating the contribution of both the phenolic content and the nanosize to the antibacterial activity. Studies on the antibacterial mode of action showed that bacteria in contact with the functionalized NPs presented decreased metabolic activity and high levels of reactive oxygen species (ROS). Moreover, PheLigNPs demonstrated affinity to the bacterial surface and the ability to cause membrane destabilization. Antimicrobial resistance studies showed that the NPs did not induce resistance in pathogenic bacteria, unlike traditional antibiotics.Peer ReviewedPostprint (published version

New myeloperoxidase detection system based on enzyme-catalysed oxidative synthesis of a dye for paper-based diagnostic devices

The severity and cost of wound infections strongly demands for simple and fast methods for wound infection determination. Point-of-care testing devices play a crucial role in order to achieve a fast diagnosis and early treatment. Myeloperoxidase (MPO) enzyme, detected in fluids of infected wounds has been postulated as a suitable biomarker for wound diagnostics. Here we present a new system for MPO detection, based on enzyme-catalysed oxidative synthesis of a dye that can be incorporated into paper-based point of care devices. Visual MPO detection has been achieved through the use of phenylenediamine, a common colourless hair dye precursor. MPO oxidation of these compounds yielded bright coloured products distinguishable from the colour of the wound environment. Immobilisation of the MPO substrates on paper strips was achieved through in situ interaction of the oxidised coloured product with branched polyethyleneimine. The colour reaction of the immobilized substrates, detectable by naked eye, responds to the MPO levels present in infected wound fluids revealing an easy system for incorporation of MPO detection in paper based diagnostic devices.Peer ReviewedPostprint (author's final draft

Electrical monitoring of infection biomarkers in chronic wounds using nanochannels

Chronic wounds represent an important healthcare challenge in developed countries, being wound infection a serious complication with significant impact on patients’ life conditions. However, there is a lack of methods allowing an early diagnosis of infection and a right decision making for a correct treatment. In this context, we propose a novel methodology for the electrical monitoring of infection biomarkers in chronic wound exudates, using nanoporous alumina membranes. Lysozyme, an enzyme produced by the human immune system indicating wound infection, is selected as a model compound to prove the concept. Peptidoglycan, a component of the bacterial layer and the native substrate of lysozyme, is immobilized on the inner walls of the nanochannels, blocking them both sterically and electrostatically. The steric blocking is dependent on the pore size (20 - 100 nm) and the peptidoglycan concentration, whereas the electrostatic blocking depends on the pH. The proposed analytical method is based on the electrical monitoring of the steric/electrostatic nanochannels unblocking upon the specific degradation of peptidoglycan by lysozyme, allowing to detect the infection biomarker at 280 ng/mL levels, which are below those expected in wounds. The low protein adsorption rate and thus outstanding filtering properties of the nanoporous alumina membranes allowed us to discriminate wound exudates from patients with both sterile and infected ulcers without any sample pre-treatment usually indispensable in most diagnostic devices for analysis of physiological fluids. Although size and charge effects in nanochannels have been previously approached for biosensing purposes, as far as we know, the use of nanoporous membranes for monitoring enzymatic cleavage processes, leading to analytical systems for the specific detection of the enzymes has not been deeply explored so far. Compared with previously reported methods, our methodology presents the advantages of no need of neither bioreceptors (antibodies or aptamers) nor competitive assays, low matrix effects and quantitative and rapid analysis at the point-of-care, being also of potential application for the determination of other protease biomarkers.Peer ReviewedPostprint (published version

Rapid colorimetric detection of wound infection with a fluidic paper device

Current procedures for the assessment of chronic wound infection are time-consuming and require complex instruments and trained personnel. The incidence of chronic wounds worldwide, and the associated economic burden, urge for simple and cheap point-of-care testing (PoCT) devices for fast on-site diagnosis to enable appropriate early treatment. The enzyme myeloperoxidase (MPO), whose activity in infected wounds is about ten times higher than in non-infected wounds, appears to be a suitable biomarker for wound infection diagnosis. Herein, we develop a single-component foldable paper-based device for the detection of MPO in wound fluids. The analyte detection is achieved in two steps: (i) selective immunocapture of MPO, and (ii) reaction of a specific dye with the captured MPO, yielding a purple color with increasing intensity as a function of the MPO activity in infected wounds in the range of 20–85 U/mL. Ex vivo experiments with wound fluids validated the analytic efficiency of the paper-based device, and the results strongly correlate with a spectrophotometric assay.Peer ReviewedPostprint (published version

Biomedical textiles

The use of textiles in medicine dates back to ancient times. In modern medicine, textile fibers can be found in a wide variety of biomedical devices from simple non-woven bandages for wounds to complex hollow fiber membranes in artificial lungs. The combination of biotechnology and textile engineering could improve the performance of biomedical textiles or produce novel textile components for biomedical devices. This chapter overviews the recent progress of enzyme biotechnology for biomedical textiles in three different aspects: i) enzymes as tools for fiber functionalization, ii) enzymes immobilized on textiles carriers for therapeutic or bioanalytical application, and iii) enzymatically responsive textiles. Innovative technologies for upgrading the functional properties of biomedical textiles are also considered.Postprint (author's final draft

Electrochemical quantification of biomarker myeloperoxidase

Point of care testing (PoCT) devices permit precise and rapid detection of disease-related biomarkers contributing to an early disease diagnosis and administration of an appropriate treatment. The enzyme myeloperoxidase (MPO) is a relevant biomarker for infection and inflammation events assessment; however its direct electrochemical quantification is hindered by the limited accessibility to the iron atom in its active center. Herein, such hindrance of the MPO biomolecule is overcome using the redox mediator 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). The charge involved in the electrochemical reduction of the MPO-oxidized ABTS is correlated with the concentration of MPO. The use of ABTS allowed for the electrochemical assessment of a wide range of MPO concentrations (10–1000 nM) including those reported for wound infections, chronic obstructive pulmonary disease and early adverse cardiac events. The developed electroanalytical approach is rapid and inexpensive, and thus suitable for implementation in PoCT devices.Peer ReviewedPostprint (author's final draft

Antibacterial coatings on medical devices

Peer ReviewedPostprint (author's final draft

Antibacterial coatings on medical devices

Peer Reviewe

Hybrid tellurium-lignin nanoparticles with enhanced antibacterial properties

The surge of antibiotic-resistant bacteria is leading to the loss of antibiotic treatment effectiveness, resulting in prolonged infections and even death. Against this healthcare threat, antimicrobial nanoparticles that hamper the evolve of resistance mechanisms are promising alternatives to antibiotics. Herein, we used Kraft lignin, a poorly valorized polymer derived from plant biomass, to develop novel hybrid tellurium-lignin nanoparticles (TeLigNPs) as alternative antimicrobial agents. The sonochemically synthesized TeLigNPs are comprised of a lignin matrix with embedded Te clusters, revealing the role of lignin as both reducing agent and structural component. The hybrid NPs showed strong bactericidal effect against the Gram-negative Escherichia coli and Pseudomonas aeruginosa, achieving more than 5 log bacteria reduction, while only slightly inhibited the growth of the Gram-positive Staphylococcus aureus. Exposure of TeLigNPs to human cells did not cause morphological changes or reduction in cell viability. Studies on the antimicrobial mechanism of action demonstrated that the novel TeLigNPs were able to disturb bacterial model membranes and generate reactive oxygen species (ROS) in Gram-negative bacteria.Peer ReviewedPostprint (published version