The spread of bacteria and infections, initially associated with an increased number of hospital- acquired infections, has now extended into the community causing severe and difficult to diagnose and treat diseases. An important preventive measure for providing bacteria-free environment for the patients is the introduction of highly efficient and durable antibacterial textiles in the hospitals. This work reports on a simultaneous sonochemical-enzymatic process for durable antibacterial coating of medical textiles with zinc oxide nanoparticles and natural compounds. The ultrasound irradiation, used for the generation of the nano-structures, and its combination with enzymes (oxidative laccase or hydrolytic cellulase) resulted in “ready-to- use” hybrid coatings on the textiles that significantly inhibited the growth of medically relevant Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. The textile materials resisted the intensive laundry regimes used in hospitals and retained their bactericidal activity even after multiple washings cycles. The use of bio-tools and the absence of harsh chemicals define the coating process as a green chemistry approach for design of durable and biocompatible medical textiles.
The increased emergence of drug resistant bacteria is one of the most serious problems in the modern medicine, and although new drugs are constantly being sought, the pace of development is slow compared to the evolution and spread of multidrug resistant species. In this study, the efficacy of the commercially available antibiotics (e.g. vancomycin and gentamicin) was synergistically enhanced by the enzymatic disruption of bacterial quorum sensing (QS) and ultrasound assisted nanotransformation of the antibacterial agents. The generated hybrid nano-antibacterials were deposited on silicone material in a Layer-by-Layer fashion. These nanocoatings disrupted bacterial QS signaling in reporter Chromobacterium violaceum and attenuated the virulence of P. aeruginosa, as demonstrated by the decrease of violacein, pyocyanin and alkaline protease production. Moreover, the multilayers eradicated Pseudomonas aeruginosa planktonic cells and inhibited up to 80 % the bacterium biofilm growth, without affecting the viability of human fibroblasts. Our results demonstrated that the hybrid nano-antibacterials, with complimentary modes of action, might be valuable alternatives to control drug-resistant biofilm occurrence on medical devices at reduced antibiotic dosages.
Bacteria-mediated diseases are a global healthcare concern due to the development and spread of antibiotic resistant strains. Cationic compounds are considered membrane active biocidal agents having a great potential to control bacterial infections, while limiting the emergence of drug resistance. Herein, the versatility and simplicity of the Layer-by- Layer (LbL) technique was used to functionalize polymer nanoparticles with antibacterial aminocellulose conjugate in a multilayer fashion. Stable polyelectrolyte-decorated particles with an average size of 250 nm and zeta potential of ± 40 mV were developed after five LbL assembly cycles. The antibacterial activity of these particles against Gram- positive Staphylococcus aureus and Gram-negative Escherichia coli increased significantly when the polycationic aminocellulose was applied as an uppermost layer. The large number of amino groups available on the particles surface improved the interaction with bacterial membrane phospholipids leading to membrane disturbance as was confirmed by Langmuir monolayer. The biopolymer decorated NPs were also able to inhibit the drug resistant biofilm formation, without affecting the human cells viability and therefore are promising alternatives for controlling bacterial infections occurrence.
Health-care associated infections (HAIs), or infections acquired in health-care facilities, affect hundreds of millions of patients worldwide each year. They are a major financial issue for the European healthcare system and were recognised as a global threat associated with medical care. The alarming statistics show that 1 in 10 patients develop infection in the hospital. About 400 000 of the HAIs are caused by antibiotic-resistant strains, estimated to cause economic loss in Europe of more than €1.5 billion.
Microbial biofilms are the reason for 80% of all infections currently treated in the hospitals. When established on catheters, heart valves, implants, and intrauterine devices the biofilms usually cause severe chronic infection, as well as systemic dissemination of the pathogen and dysfunction of the device. Catheter-associated urinary tract infections (CAUTIs) are the most frequent HAI worldwide that account for over 40% of all nosocomial infections and form 80% of all urinary tract infections in the hospitals. They are global health issue that delays the patient’s recovery and significantly increases healthcare costs, length of antibiotic therapy and risk of resistance development.
Microbiologically unsafe water is among the major causes of preventable morbidity and mortality. The World Health Organisation (WHO) reports that by 2025 half of the world’s population will be living in water scarcity areas with access only to inadequate drinking water leading to bacteria waterborne diseases such as diarrhea, cholera, dysentery and typhoid fever. Annually 2.2 million diarrheal disease deaths are being linked with the consumption of contaminated water. The extreme rainfall and flooding, as a result of the global warming, contribute to the microbial proliferation in surface and groundwater.
Antibiotic resistance is a worldwide problem. The emergence of antibiotic resistant bacteria is one of the most serious health threats nowadays. Efforts to prevent such threat are based on infection control in hospital and public settings and prevention of person-to-person spread. As a part of the public health strategy against the microbial infections, PROTECT will develop a versatile platform of 3 pre-commercial lines for production of antimicrobial textiles for hospitals and public areas, and anti-biofilm medical devices and water treatment membranes. These production lines will share as a common feature the use of high intensity ultrasound (US) in 3 different machinery designs: continuous roll-to-roll (R2R) US coating; continuous R2R spray coating with US nozzles, and batch mode (non-continuous) US coating.
Perez, S.; Pashkuleva, I.; Gedanken, A.; Vidal, F.; Wey, M.; Sousa, R.A.; Tzanov, T. European & Global Summit for Clinical Nanomedicine and Targeted Medicine p. 200-201 Data de presentació: 2017-05-08 Presentació treball a congrés
Onestep enzymatic crosslinking approach was used to synthesize multifunctional hydrogels for chronic wounds from thiolated chitosan and gallic acid. Stable at physiological conditions hydrogels with tunable physicomechanical and functional properties were generated with the aid of the oxidative enzyme laccase. The potential of these hydrogels as wound dressing materials was evaluated in vitro against major factors governing the chronicity in wounds, such as myeloperoxidase (MPO) and matrix metalloproteinases (MMPs) activities, reactive oxygen species (ROS) and bacterial contamination. The study revealed a decrease of the MPO and collagenase activities by up to 98 % and 23 %, respectively. The hydrogels showed significant antioxidant activity, scavenging the free 1,1diphenyl2picrylhydrazyl radical up to 94 %. In addition, the hydrogels reduced by 2 logs the bacterial population of both S.aureus and E.coli strains. Exvivo studies with a wound exudate collected from a patient with a venous leg ulcer confirmed the inhibitory capacity of the hydrogels against MPO and MMPs. The hydrogels showed up to 99 % biocompatibility over 24 and 168 hours of incubation. This new dressing material combines in a unique design several beneficial to wound healing properties – control over the MMP/MPO activity, antioxidant and antimicrobial effect, coupled to high swelling capacity for exudate retention
Bacteriamediated diseases are global healthcare concern due to the accelerated emergence and spread of drug resistant bacterial strains. Cationic compounds are considered membrane active biocidal agents having a great potential to control bacterial infections while limiting the emergence of drug resistance. Herein, the versatility and simplicity of the LayerbyLayer (LbL) technique was used to functionalize poly (methyl vinyl ethercomaleic anhydride) nanoparticles with a highly antibacterial aminocellulose conjugate in a multilayer fashion. The assembly of cationic bearing aminocellulose with negatively charged hyaluronic acid on the particles surface was confirmed by the alternations of their surface charge and size after each deposition step. Stable polyelectrolyte decorated particles with an average size of 600 nm and zeta potential of ± 40 mV were developed after five LbL assembly cycles. The antibacterial properties of these particles against S. aureus were significantly improved when the polycationic aminocellulose was applied as a top layer. The large number of amino groups available on the particles surface allows for the interaction with intrinsically anionic bacterial cell wall leading to irreparable membrane damage and complete eradication of S. aureus after 24 h treatment, while the hyaluronic acid improve their biocompatibility. This specific mechanism of action is believed to diminish the possibility for selection of new resistant strains and is a great promise in controlling bacterial contamination
Bacterial infections caused by drug resistant strains are one of the world’s public health challenges and the development of alternative strategies is extensively being searched. Vancomycin is a glycopeptide antibiotic largely used for treatment of infections caused by Grampositive bacteria. However, it has little effect against Gramnegative bacteria due to its inability to penetrate their outer membrane, impermeable to such large molecules. The assemblies of molecules at nanometer dimensions confers unique properties, differing from those of the free molecules and the bulk materials with the same composition. The nanotransformation of the active agents themselves into nanosize has been applied in our group using the ultrasonic emulsification method to synthesize oilfilled nanospheres (NSs), in which the antimicrobial agent is located at the interface of the droplet. The nanotransformation does not alter the target of the drug or its chemical structure, while simply adding a support mechanism to its mode of action towards bacteria. This mode of action reduces the possibility of developing new resistant strains because bacterial membrane is highly evolutionarily conserved. The observed penetration of the NSs within a Langmuir monolayer composed by bacterial membrane phospholipids has confirmed that the vancomycin nanospheres were able to interact with the membrane and affect the physical integrity of Escherichia coli cells, which was further visualized by scanning electron microscopy. The ability of Vancomycin NSs to kill biofilm was visualised using fluorescence microscopy and antibacterial assays and quantified using CBDMBEC™ technique. The nanospherization of vancomycin boosted its capacity to inhibit the growth of Gramnegative Escherichia coli and Pseudomonas aeruginosa , making these bacteria up to 10 and 100 times, respectively, more sensitive to the antibiotic. Moreover, the antibiotic nanospheres eradicated biofilms of Gramnegative bacteria in the antibacterialeffective concentration
Zwitterionic polymers (ZP) recently emerged as biomaterials with excellent bio and haemo compatibility. The protein adsorption on their surfaces is lower even compared to the golden standard in the field – poly(ethylene glycol). Moreover, ZP are known to swell more in salt solutions than in pure water, which is known as the antipolyelectrolyte effect. Thus, the ZP combine antifouling properties with high ability to absorb wound exudate and at the same time maintain the moisture environment of chronic wound. Nevertheless, their potential for chronic wound management is still unrevealed. In this work, two types of ZP networks were synthesized comprising in their structure zwitterionic moieties. Both ZP types are saltsensitive. In addition, polysulfobetain (PSB) is thermoresponsive, while polycarboxybetain (PCB) is pH sensitive, allowing for modulation of their structure and functional properties by external stimuli. The PSB and PCB hydrogels were characterized in terms of their physicomechanical properties, biocompatibility and inhibitory activities towards bacterial biofilms and major enzymes causing chronicity of the wounds, such as myeloperoxidase and matrix metalloproteinases. The potential of the hydrogels for chronic wound treatment was confirmed by ex vivo experiments with clinical exudates.
Zille, A.; Fernandes, M.; Francesko, A.; Tzanov, T.; ; Oliveira, F.; Almeida, L.; Souto, A.; Carneiro, N.; Amorim, T. American Chemical Society National Meeting & Exposition Data de presentació: 2017-04-02 Presentació treball a congrés
Tzanov, T.; Petkova, P.; Stefanov, I.; Francesko, A.; Díaz, C.; Aracri, E. International Conference on Fiber and Polymer Biotechnology p. 21 Data de presentació: 2016-09-08 Presentació treball a congrés
Infection is a global problem. The resulting pain, impairment and social isolation lead to reduced quality of life and, in the worst case, hospitalization, and eventually sepsis and death. Infected wound fluids contain several enzymes, produced by the cells involved in the immune response, which can be used as markers of infection. Myeloperoxidase (MPO) is one of the enzymes present in the white blood cells and implicated in the first response of the immune system upon inflammation or injury. MPO substrates are phenolic molecules developing color upon oxidation by this enzyme.
Point of care (PoC) devices are wellknown for the fast detection of hormones and metabolites associated to diseases or other body conditions. Here we show the initial steps to build up a PoC gadget to detect MPO levels after injury following two different strategies. In a first approach, several substrates for MPO, chosen by the distinguishable products obtained upon reaction, have been immobilized on a nitrocellulose membrane. In a second approach, a specific antibody has been coated on the membrane and upon enzyme binding; MPO is detected by the addition of the substrates. Infected and noninfected wounds activity levels of MPO have been tested on both approaches.
Our results show that MPO wicks on the membrane and reacts with the immobilized substrates and binds to the antibody keeping its activity. High activities of MPO showed a characteristic change of color on the test area while lower activities were undetectable. However, MPO wicking is not uniformly distributed through the membrane and new strategies for improvement are needed on this matter.
The development of this device will help to distinguish between infected and noninfected wounds reducing unnecessary antibiotic administration and consequently decrease antibiotic resistance.
Freestanding membranes assembled from polyelectrolyte multilayers are promising new materials since they offer the possibility of engineering their chemical and mechanical properties. Herein, the buildup of biocompatible freestanding multilayer membranes consisting in biopolymers/silver nanoparticles (Ag NPs) and hyaluronic acid (HA) is accomplished using layer by layer (LbL) technique. To obtain the biopolymer/Ag NPs, solventfree synthesis by chemical reduction of Ag NPs is carried out in presence of chitosan (CS) or 6deoxy6(2aminoethyl) aminocellulose (AC) leading to simultaneous NPs biofunctionalization. The use of the aminofunctional biopolymers as doping agents prevents the NPs aggregation and thus, results in stable dispersions of Ag NPs. Further, based on electrostatic interactions, LbL selfassembly is carried out to prepare freestanding nanocomposite membranes (consisted of 100 and 200 bilayers) of hybrid CS(AC)/Ag NPs and HA. The alternating monolayers of positively charged aminofunctionalized Ag NPs and negatively charged HA are sequentially assembled on a silicone substrate. The capability of the resulting multilayer membranes to prevent the biofilm formation and to inhibit the bacterial growth of Gramnegative Escherichia coli and Grampositive Staphylococcus aureus bacteria is demonstrated. Moreover, the good biocompatibility of the obtained nanocomposite membranes with human fibroblast cell line suggested their suitability for biomedical applications such as wound healing material.
The increasing prevalence of bacterial strains with resistance to conventional antibiotics is a major global healthcare problem. Strategies based on the cellular response of bacteria to antimicrobial agents have been followed to reduce the adaptive pressure on these bacteria. One of these strategies consists in damaging the bacterial membrane, which leads to leakage of intracellular constituents into the extracellular environment and changes in intracellular pH. Langmuir technique has been used to evaluate the interaction of several antibacterial agents with cell membrane models at the molecular level.
In this work Vancomycin (VAC) a widely used antibiotic with acquired bacteria resistance – was transformed sonochemically into nanoparticles (NPsVAC) which antibacterial efficiency was further evaluated on biomimetic bacterial membranes. The results indicate a higher membraneVAC interaction when antibiotic is in nanoform rather than in solution.
On the other hand, nanoparticles (NPs) during circulation in biological fluids are affected by the protein corona phenomenon that consists in the adsorption of biomolecules on the NPs surface altering thereby their targeting and efficiency. Thus, the bacterial membraneNPsVAC interactions were also studied in corona effect conditions, in order to simulate the real application scenario.
The increasing emergence of multidrug resistant bacterial strains is one of the most serious problems in modern medicine. To overcome the action of conventional antibiotics bacteria develop various effective resistance mechanisms including the formation of surfaceattached communities of cells enclosed in extracellular polymeric matrix, known as biofilms. Biofilms formed on indwelling medical devices cause life threatening infections and are associated with increased mortality and morbidity in the hospitals. Therefore, novel strategies for prevention and eradication of drug resistant bacterial biofilms have been sought. This work reports the development of antibacterial and antibiofilm coatings on medical devices comprising polyphenols and antibiotic nanocapsules (NCs). The widely used antibiotic gentamicin was first sonochemically processed into NCs improving its antibacterial potential. Afterwards, the nanoantibiotic was combined with the antioxidant tannic acid to build bacteria resistant multilayer coatings on the surface of silicone urinary catheters. To improve the stability of the coatings at physiological conditions, the deposited layers were further crosslinked with laccase enzyme. The antibacterial efficiency of the coatings with nano gentamicin was significantly enhanced compared to the coatings of nonprocessed antibiotic. Moreover, the biofilm formation of clinically relevant Gramnegative Pseudomonas aeruginosa and Grampositive Staphylococcus aureus was reduced by 40 % in static conditions. The enhanced antibiofilm activity of the nanoantibiotic assemblies was also demonstrated in vitro under dynamic conditions using a model of catheterized human bladder. Laccaseassisted crosslinking of the layers resulted in the formation of stable coatings able to counteract biofilm occurrence over seven days of catheterization.
Nosocomial infections are the infections that patients acquired in hospitals. These infections represent a leading cause of death, increased stay in hospitals and healthcare costs. Here we describe several antimicrobial approaches for biotechnical functionalisation of medical textiles and indwelling devices, and the early diagnosis of infection.
Medical textiles coated with antimicrobial nanoparticles (NPs). Current technologies for antimicrobial NPs coating of medical textiles are characterised by low coating stability and poor uniformity of the coatings, leaching of NPs, energyconsuming coating processes and dark colour of silver – the most widely used antimicrobial agent. Alternatively, sonochemistry can be used as a versatile tool for designing of antimicrobial surfaces. Combined sonoenzymatic coating of fabrics with ZnO improved the NPs uniformity and adhesion, and enhanced their antimicrobial activity. The antibacterial efficiency of the coatings resisted multiple washing cycles at hospital laundering regimes.
Antibiofilm indwelling medical devices. 80% of all urinary tract infections treated in hospitals are due to biofilm formation on the catheters. Building a dual antimicrobial/antifouling coating on silicone urinary catheters using an enzymetriggered bottomup approach efficiently prevented the biofilm formation of both Gram+ and Gram bacteria. Bacterial cell–tocell communication, involved in biofilm formation, is regulated through secretion and uptake of extracellular signals called autoinducers (AIs). Coating of urinary catheters with enzymes quenching the AIs and degrading the biofilm matrix provided efficient antibiofilm effect both in vitro and in vivo. Importantly, the enzymatic quenching of AIs was achieved in the extracellular environment eluding the intrinsic resistancedevelopment mechanisms of bacteria and attenuating bacterial virulence. Moreover, the nanoformulation of these enzymes with clinically relevant antibiotics provided increased bacteria susceptibility at lower antibiotic dosage. Enzymes can be used as biomarkers for wound infection in a diagnostic kit. This kit relies on the detection of overexpressed during infection MMPs, MPO and lysozyme, implementing two complementary strategies to detect bacterial infection immunochemical quantification of the enzyme proteins, and quantification of the enzymatic activities.
Bacterial biofilms are formed when unicellular organisms come together to form a community that is attached to a solid surface and encased in an exopolysacharide matrix. When growing in the biofilm phenotype, bacteria are able to survive in hostile environments and acquire increased antibiotic tolerance and resistance to clearance by the host immune system. Currently there is an urgent need for new antibacterial agents with low susceptibility to resistance development. In this study, two conventional antibiotics, to which pathogenic bacteria such as Pseudomonas aeruginosa and Escherichia coli are resistant, were processed into nanospheres using a one-step, environmentally friendly sonochemical technology. The penicillin G and vancomycin nanospheres were shown to possess improved antibacterial and antibiofilm activity compared to the non-processed antibiotics. Their effect was further related to the enhanced membrane permeability of the spheres, studied by their interaction with cell membrane models - Langmuir monolayers - allowing minimum diffusion limitations and maximum surface area per unit mass. Importantly, the studied nano-structured materials selectively killed bacteria, without imparting toxicity to human cells. It is believed that bacteria do not recognize antibiotic nanospheres as a threat, hence their efficiency is improved and a delay in the development of bacteria resistance mechanisms probable.
Bacteria that grow in complex biofilm communities embedded in a self-produced extracellular polymeric matrix are a global health concern. When these microorganisms colonize indwelling medical devices they cooperate through a quorum sensing mechanism and form well-established biofilms that resist antibiotic treatments and cause difficult to treat infections. Despite the recent advances in antimicrobial research, efficient antibiofilm strategies for control of bacteria adhesion and proliferation on the surfaces of medical devices are still needed. In this study, quorum quenching acylase and exopolysaccharide-degrading a-amylase enzymes were deposited on silicone urinary catheters in a layer-by-layer fashion in order to investigate their potential to prevent bacterial biofilm formation. The assembling of the hybrid multilayer coatings on the catheter surface was achieved by alternate deposition of negatively charged enzymes and positively charged polyethylenimine. The hybrid coatings simultaneously interfered with the bacterial quorum sensing and degraded the biofilm polysaccharide matrix, enhancing up to 30 % the inhibition of Pseudomonas aeruginosa biofilm formation when compared to the enzymes applied individually on the silicone material. Furthermore, the dual-species biofilm occurrence (Pseudomonas aeruginosa and Escherichia coli) on silicone urinary catheters was significantly reduced under dynamic conditions in an in vitro catheterized bladder model. The developed antibiofilm coatings did not cause toxicity on the human fibroblasts cell line (BJ-5ta) over seven days and thus constitute a viable alternative to control bacterial biofilms on urinary catheters.
Macedo, M.M.; Francesko, A.; Torrent, J.; Torrent-Burgués, J.; Heinze, T.; Tzanov, T. International Conference on Polymer and Fiber Biotechnology Data de presentació: 2014-05-25 Presentació treball a congrés
Nanoparticles possess unique physicochemical properties, important for the efficient transport through the cell membranes. The small and controllable size, large surface area to mass ratio, and high reactivity facilitate the intracellular delivery, thereby overcoming some of the limitations in traditional antimicrobial therapeutics. Chemically modified biopolymers (thiolated chitosan and aminocellulose) were processed into nanoparticles via a one-step sonochemical process and evaluated for their antibacterial activity against Escherichia coli and Staphyloccocus aureus. Thiolated chitosan and aminocellulose were shown to possess improved antimicrobial properties compared to the starting biopolymers and even higher bactericide effect was observed after processed into nanoparticles. The mechanistic insights were obtained by Langmuir monolayer technique using Escherichia coli phospholipids as membrane models. The high cationic character of the modified biopolymers and the obtained spherical structure were found to allow efficient interaction with the phospholipid heads and tails, crucial for their antibacterial activity and transport through cell membranes.