Microwave-Assisted Hydrothermal Treatment of Multifunctional Substituted Hydroxyapatite with Prospective Applications in Bone Regeneration.

Orthopedic bone graft infections are major complications in today's medicine, and the demand for antibacterial treatments is expanding because of the spread of antibiotic resistance. Various compositions of hydroxyapatite (HAp) in which Calcium (Ca2+) ions are substituted with Cerium (Ce3+) and Magnesium (Mg2+) are herein proposed as biomaterials for hard tissue implants. This approach gained popularity in recent years and, in the pursuit of mimicking the natural bone mineral's composition, over 70 elements of the Periodic Table were already reported as substituents into HAp structure. The current study aimed to create materials based on HAp, Hap-Ce, and Hap-Mg using hydrothermal maturation in the microwave field. This route has been considered a novel, promising, and effective way to obtain monodisperse, fine nanoparticles while easily controlling the synthesis parameters. The synthesized HAp powders were characterized morphologically and structurally by XRD diffraction, Dynamic light scattering, zeta potential, FTIR spectrometry, and SEM analysis. Proliferation and morphological analysis on osteoblast cell cultures were used to demonstrate the cytocompatibility of the produced biomaterials. The antimicrobial effect was highlighted in the synthesized samples, especially for hydroxyapatite substituted with cerium. Therefore, the samples of HAp substituted with cerium or magnesium are proposed as biomaterials with enhanced osseointegration, also having the capacity to reduce device-associated infections.

Silver nanoparticles decorated ZnO-CuO core-shell nanowire arrays with low water adhesion and high antibacterial activity.

Nanostructured surfaces based on silver nanoparticles decorated ZnO-CuO core-shell nanowire arrays, which can assure protection against various environmental factors such as water and bacteria were developed by combining dry preparation techniques namely thermal oxidation in air, radio frequency (RF) magnetron sputtering and thermal vacuum evaporation. Thus, high-aspect-ratio ZnO nanowire arrays were grown directly on zinc foils by thermal oxidation in air. Further ZnO nanowires were coated with a CuO layer by RF magnetron sputtering, the obtained ZnO-CuO core-shell nanowires being decorated with Ag nanoparticles by thermal vacuum evaporation. The prepared samples were comprehensively assessed from morphological, compositional, structural, optical, surface chemistry, wetting and antibacterial activity point of view. The wettability studies show that native Zn foil and ZnO nanowire arrays grown on it are featured by a high water droplet adhesion while ZnO-CuO core-shell nanowire arrays (before and after decoration with Ag nanoparticles) reveal a low water droplet adhesion. The antibacterial tests carried on Escherichia coli (a Gram-negative bacterium) and Staphylococcus aureus (a Gram-positive bacterium) emphasize that the nanostructured surfaces based on nanowire arrays present excellent antibacterial activity against both type of bacteria. This study proves that functional surfaces obtained by relatively simple and highly reproducible preparation techniques that can be easily scaled to large area are very attractive in the field of water repellent coatings with enhanced antibacterial function.

Antimicrobial Efficiency of Some Essential Oils in Antibiotic-Resistant Pseudomonas aeruginosa Isolates.

Pseudomonas aeruginosa is a non-fermentative Gram-negative opportunistic pathogen, frequently encountered in difficult-to-treat hospital-acquired infections and also wastewaters. The natural resistance of this pathogen, together with the frequent occurrence of multidrug-resistant strains, make current antibiotic therapy inefficient in treating P. aeruginosa infections. Antibiotic therapy creates a huge pressure to select resistant strains in clinical settings but also in the environment, since high amounts of antibiotics are released in waters and soil. Essential oils (EOs) and plant-derived compounds are efficient, ecologic, and sustainable alternatives in the management of various diseases, including infections. In this study, we evaluated the antibacterial effects of four commercial essential oils, namely, tea tree, thyme, sage, and eucalyptus, on 36 P. aeruginosa strains isolated from hospital infections and wastewaters. Bacterial strains were characterized in terms of virulence and antimicrobial resistance. The results show that most strains expressed soluble pore toxin virulence factors such as lecithinase (89-100%) and lipase (72-86%). All P. aeruginosa strains were positive for alginate encoding gene and 94.44% for protease IV; most of the strains were exotoxin producers (i.e., 80.56% for the ExoS gene, 77.78% for the ExoT gene, while the ExoU gene was present in 38.98% of the strains). Phospholipase-encoding genes (plc) were identified in 91.67/86.11% of the cases (plcH/plcN genes). A high antibiotic resistance level was identified, most of the strains being resistant to cabapenems and cephalosporins. Cabapenem resistance was higher in hospital and hospital wastewater strains (55.56-100%) as compared to those in urban wastewater. The most frequently encountered encoding genes were for extended spectrum ß-lactamases (ESBLs), namely, blaCTX-M (83.33% of the strains), blaSHV (80.56%), blaGES (52.78%), and blaVEB (13.89%), followed by carbapenemase-encoding genes (blaVIM, 8.33%). Statistical comparison of the EOs' antimicrobial results showed that thyme gave the lowest minimum inhibitory concentrations (MIC) and minimum biofilm eradication concentrations (MBEC) in P. aeruginosa-resistant isolates, making this EO a competitive candidate for the development of efficient and ecologic antimicrobial alternatives.

Modulation of Gut Microbiota by Essential Oils and Inorganic Nanoparticles: Impact in Nutrition and Health.

Microbiota plays a crucial role in human health and disease; therefore, the modulation of this complex and yet widely unexplored ecosystem is a biomedical priority. Numerous antibacterial alternatives have been developed in recent years, imposed by the huge problem of antibioresistance, but also by the people demand for natural therapeutical products without side effects, as dysbiosis, cyto/hepatotoxicity. Current studies are focusing mainly in the development of nanoparticles (NPs) functionalized with herbal and fruit essential oils (EOs) to fight resistant pathogens. This is due to their increased efficiency against susceptible, multidrug resistant and biofilm embedded microorganisms. They are also studied because of their versatile properties, size and possibility to ensure a targeted administration and a controlled release of bioactive substances. Accordingly, an increasing number of studies addressing the effects of functional nanoparticles and plant products on microbial pathogens has been observed. Regardless the beneficial role of EOs and NPs in the treatment of infectious diseases, concerns regarding their potential activity against human microbiota raised constantly in recent years. The main focus of current research is on gut microbiota (GM) due to well documented metabolic and immunological functions of gut microbes. Moreover, GM is constantly exposed to micro- and nano-particles, but also plant products (including EOs). Because of the great diversity of both microbiota and chemical antimicrobial alternatives (i.e., nanomaterials and EOs), here we limit our discussion on the interactions of gut microbiota, inorganic NPs and EOs. Impact of accidental exposure caused by ingestion of day care products, foods, atmospheric particles and drugs containing nanoparticles and/or fruit EOs on gut dysbiosis and associated diseases is also dissected in this paper. Current models developed to investigate mechanisms of dysbiosis after exposure to NPs/EOs and perspectives for identifying factors driving EOs functionalized NPs dysbiosis are reviewed.

Preliminary Study on Light-Activated Antimicrobial Agents as Photocatalytic Method for Protection of Surfaces with Increased Risk of Infections.

Preventing and controlling the spread of multidrug-resistant (MDR) bacteria implicated in healthcare-associated infections is the greatest challenge of the health systems. In recent decades, research has shown the need for passive antibacterial protection of surfaces in order to reduce the microbial load and microbial biofilm development, frequently associated with transmission of infections. The aim of the present study is to analyze the efficiency of photocatalytic antimicrobial protection methods of surfaces using the new photocatalytic paint activated by light in the visible spectrum. The new composition is characterized by a wide range of analytical methods, such as UV-VIS spectroscopy, electron microscopy (SEM), X-ray powder diffraction (PXRD) or X-ray photoelectron spectroscopy (XPS). The photocatalytic activity in the UV-A was compared with the one in the visible light spectrum using an internal method developed on the basis of DIN 52980: 2008-10 standard and ISO 10678-2010 standard. Migration of metal ions in the composition was tested based on SR EN1186-3: 2003 standard. The new photocatalytic antimicrobial method uses a type of photocatalytic paint that is active in the visible spectral range and generates reactive oxygen species with inhibitory effect against all tested microbial strains.

Modulation of Quorum Sensing and Biofilms in Less Investigated Gram-Negative ESKAPE Pathogens.

Pathogenic bacteria have the ability to sense their versatile environment and adapt by behavioral changes both to the external reservoirs and the infected host, which, in response to microbial colonization, mobilizes equally sophisticated anti-infectious strategies. One of the most important adaptive processes is the ability of pathogenic bacteria to turn from the free, floating, or planktonic state to the adherent one and to develop biofilms on alive and inert substrata; this social lifestyle, based on very complex communication networks, namely, the quorum sensing (QS) and response system, confers them an increased phenotypic or behavioral resistance to different stress factors, including host defense mechanisms and antibiotics. As a consequence, biofilm infections can be difficult to diagnose and treat, requiring complex multidrug therapeutic regimens, which often fail to resolve the infection. One of the most promising avenues for discovering novel and efficient antibiofilm strategies is targeting individual cells and their QS mechanisms. A huge amount of data related to the inhibition of QS and biofilm formation in pathogenic bacteria have been obtained using the well-established gram-positive Staphylococcus aureus and gram-negative Pseudomonas aeruginosa models. The purpose of this paper was to revise the progress on the development of antibiofilm and anti-QS strategies in the less investigated gram-negative ESKAPE pathogens Klebsiella pneumoniae, Acinetobacter baumannii, and Enterobacter sp. and identify promising leads for the therapeutic management of these clinically significant and highly resistant opportunistic pathogens.

Eugenol-Functionalized Magnetite Nanoparticles Modulate Virulence and Persistence in Pseudomonas aeruginosa Clinical Strains.

Efficient antibiotics to cure Pseudomonas aeruginosa persistent infections are currently insufficient and alternative options are needed. A promising lead is to design therapeutics able to modulate key phenotypes in microbial virulence and thus control the progression of the infectious process without selecting resistant mutants. In this study, we developed a nanostructured system based on Fe3O4 nanoparticles (NPs) and eugenol, a natural plant-compound which has been previously shown to interfere with microbial virulence when utilized in subinhibitory concentrations. The obtained functional NPs are crystalline, with a spherical shape and 10-15 nm in size. The subinhibitory concentrations (MIC 1/2) of the eugenol embedded magnetite NPs (Fe3O4@EUG) modulate key virulence phenotypes, such as attachment, biofilm formation, persister selection by ciprofloxacin, and the production of soluble enzymes. To our knowledge, this is the first report on the ability of functional magnetite NPs to modulate P. aeruginosa virulence and phenotypic resistance; our data highlights the potential of these bioactive nanostructures to be used as anti-pathogenic agents.

Antibiotic resistance profiles in cultivable microbiota isolated from some romanian natural fishery lakes included in Natura 2000 network.

BACKGROUND: The present study aims the characterization of antibiotic resistance phenotypes and encoding genes in bacterial strains isolated from some Romanian aquatic fishery lowland salted lakes. MATERIAL/METHODS: This study was conducted on 44 bacterial strains, mainly belonging to species used as microbiological indicators of fecal pollution isolated from four natural fishery lakes. All strains were tested for their antibiotic susceptibility by disk diffusion method. Simplex and multiplex PCR were performed to identify the ß-lactams antibiotic resistance genes (blaNMD, blaOXA-48, blaVIM, blaIMP, blaCTX-M, blaTEM), sulfonamides (Sul1, Sul2), tetracyclines (TetA, TetB, TetC, TetD, TetM), aminoglycosides (aac3Ia), vancomycin (VanA, VanB, VanC), macrolides (ermA, ermB, ermC) as well as the plasmid-mediated quinolone resistance (PMQR) markers (QnrA, QnrB, QnrS), and class 1 integrons (Int1, drfA1-aadA1). RESULTS: The Enterococcus spp. isolates exhibited phenotypic resistance to vancomycin (35 %) and macrolides (erythromycin) (75 %); from the vancomycin - resistant strains, 5 % harboured VanA (E. faecalis), while the erythromycin resistant isolates were positive for the ermA gene (E. faecalis - 10 %, E. faecium - 5 %). The Gram- negative rods (GNR) exhibited a high level of resistance to ß-lactams: cefuroxime (63 %), cefazolin (42 %), ceftriaxone (8 %), ceftazidime and aztreonam (4 % each). The genetic determinants for beta-lactam resistance were represented by blaCTX-M-like (33 %), blaNDM-like and blaIMP-like (8.33 %) genes. The resistance to non-ß-lactam antibiotics was ascertained to the following genes: quinolones (QnrS - 4.16 %); sulfonamides (Sul1-75 %, Sul2-4.16 %); aminoglycosides (aac3Ia - 4.16 %); tetracyclines (tetA - 25 %, tetC - 15 %). The integrase gene was found in more than 50 % of the studied strains (58.33 %). CONCLUSIONS: The cultivable aquatic microbiota from fishery lakes is dominated by enterococci and Enterobacterales strains. The GNR strains exhibited high levels of ß-lactam resistance mediated by extended spectrum beta-lactamases and metallo-ß-lactamases. The Enterococcus sp. isolates were highly resistant to macrolides and vancomycin. The high level and diversity of resistance markers, correlated with a high frequency of integrons is suggesting that this environment could act as an important reservoir of antibiotic resistance genes with a great probability to be horizontally transmitted to other associated species from the aquatic sediments microbiota, raising the potential zoonotic risk for fish consumers.

Functionalization of eggshell membranes with CuO-ZnO based p-n junctions for visible light induced antibacterial activity against Escherichia coli.

Biopolymers provide versatile platforms for designing naturally-derived wound care dressings through eco-friendly pathways. Eggshell membrane (ESM), a widely available, biocompatible biopolymer based structure features a unique 3D porous interwoven fibrous protein network. The ESM was functionalized with inorganic compounds (Ag, ZnO, CuO used either separately or combined) using a straightforward deposition technique namely radio frequency magnetron sputtering. The functionalized ESMs were characterized from morphological, structural, compositional, surface chemistry, optical, cytotoxicity and antibacterial point of view. It was emphasized that functionalization with a combination of metal oxides and exposure to visible light results in a highly efficient antibacterial activity against Escherichia coli when compared to the activity of individual metal oxide components. It is assumed that this is possible due to the fact that an axial p-n junction is created by joining the two metal oxides. This structure separates into components the charge carrier pairs promoted by visible light irradiation that further can influence the generation of reactive oxygen species which ultimately are responsible for the bactericide effect. This study proves that, by employing inexpensive and environmentally friendly materials (ESM and metal oxides) and fabrication techniques (radio frequency magnetron sputtering), affordable antibacterial materials can be developed for potential applications in chronic wound healing device area.

Impact of Pseudomonas aeruginosa quorum sensing signaling molecules on adhesion and inflammatory markers in endothelial cells.

Pseudomonas aeruginosa relies on the quorum sensing (QS) signaling system as a central regulator mechanism of virulence expression that contributes to the formation and maintenance of biofilms and tolerance to conventional antimicrobials. QS Signaling molecules (QSSMs) may be recognized and may function also within the host cells, being potentially involved in the progression of the infectious process. In this study we evaluate the expression of adhesion and inflammatory molecules in endothelial cells treated with P. aeruginosa QSSMs, in order to bring new insights on the mechanisms involved in the interaction of P. aeruginosa with host cells during the infectious process. Endothelial cells were stimulated with 20 µM of main P. aeruginosa QSSMs (OdDHL = N-(3-oxododecanoyl)-L-homoserine lactone, C4HSL = N-butyryl-L-homoserine lactone, PQS = 2-heptyl-3-hydroxy-4(1H)-quinolone and HHQ = 2-heptyl-4-quinolone). Adherence to endothelial cells, inert substratum and biofilm formation was evaluated. The expression of adhesion molecules (VE-cadherin, PECAM-1, ICAM-1, and P-selectin) and inflammatory response molecules (IL-1ß, IL-6, TNFα, TGFß, and eNOS) was assessed by qRT-PCR and flow cytometry. Our results showed that bacterial adherence to inert substratum and biofilm were decreased in the presence of all tested QSSMs. The adherence index of PAO1 laboratory strain to host cells was decreased between 10-40% in the presence of QSSMs, as compared to untreated control. Expression of eukaryotic cells adhesion molecules ICAM-1 and P-selectin was stimulated by QSSMs, whereas VE-cadherin and PECAM-1 levels were increased only by C4HSL. The inflammatory response of endothelial cells was also modulated, as observed by the modified expression of IL-1ß (for C4HSL, PQS and HHQ), IL-6 (for C4HSL and HHQ), TNFα (for C4HSL and HHQ), TGFß, and eNOS factors. Our results demonstrate that the main pseudomonadal QSSMs differentially modulate endothelial cells adhesion and proinflammatory cytokine expression. These observations provide new insights in the mechanisms by which different QSSMs activate endothelial cells and modulate the infectious process, and support the importance of recent studies aiming to develop anti-QS therapeutic strategies to fight against P. aeruginosa infections.

Aspects of Gut Microbiota and Immune System Interactions in Infectious Diseases, Immunopathology, and Cancer.

The microbiota consists of a dynamic multispecies community of bacteria, fungi, archaea, and protozoans, bringing to the host organism a dowry of cells and genes more numerous than its own. Among the different non-sterile cavities, the human gut harbors the most complex microbiota, with a strong impact on host homeostasis and immunostasis, being thus essential for maintaining the health condition. In this review, we outline the roles of gut microbiota in immunity, starting with the background information supporting the further presentation of the implications of gut microbiota dysbiosis in host susceptibility to infections, hypersensitivity reactions, autoimmunity, chronic inflammation, and cancer. The role of diet and antibiotics in the occurrence of dysbiosis and its pathological consequences, as well as the potential of probiotics to restore eubiosis is also discussed.

Development of Scaffolds for Vascular Tissue Engineering: Biomaterial Mediated Neovascularization.

Cardiovascular diseases remain the leading cause of mortality or disabled quality of life for people over the world. The necessity of neovascularization is essential for re-establishing the tissue functions after a major lesion that occurs in patients with cardiovascular disorders, such as ischemia, atherosclerosis, diabetes, peripheral vascular disease and burn wounds. This review focuses on the recent data regarding the polymers and scaffolds that are used for improving neovascularization with emphasis on the biocompatibility and mechanisms involved in stem cells proliferation, migration, adherence, differentiation and organization in vascular networks. The newly emerging techniques used in conjugation of synthetic polymers with polysaccharides or proteins attempt to improve the biocompatibility of scaffolds, but the complex structures of blood vessels make their construction to remain a major challenge for the vascular tissue engineering.

Virulence and resistance features of Pseudomonas aeruginosa strains isolated from chronic leg ulcers.

BACKGROUND: The purpose of this study was to evaluate the virulence profiles of Pseudomonas aeruginosa clinical strains recently isolated from patients hospitalized for chronic leg ulcers in the Dermatology Department of Central Military Emergency University Hospital "Carol Davila", Bucharest, Romania. METHODS: The phenotypic screening evaluated eight soluble virulence factors (haemolysins, lecithinase, lipase, caseinase, gelatinase, amylase, DNase, aesculin hydrolysis), as well as adherence ability (Cravioto adapted method) and invasion capacity on HeLa cells (gentamicin protection assay). Seven virulence genes encoding for protease IV, 3 exoenzymes (exoS, exoT, exoU), two phospholipases plcH- haemolytic phospholipase C and plcN- non-haemolytic phospholipase C) and alginate were investigated by PCR. RESULTS: The pore forming toxins and enzymes were expressed in variable proportions, the majority of the tested strains producing beta haemolysin (92.3 %), lipase (76.9 %) and lecithinase (61.5 %). The most frequent virulence genes detected in the analyzed strains were the ExoT (100 %) and AlgD (92.3 %) genes, genes codifying for phospholipases (84.6 % each of them) and for protease IV (61.5 %). CONCLUSIONS: This study reveals that correlating virulence profiles and infection clinical outcome is very useful for setting up efficient preventive and therapeutic procedures for hospitalized patients with chronic leg ulcers and positive P. aeruginosa cultures.

Efficiency of gentamicin loaded in bacterial polysaccharides microcapsules against intracellular Gram-positive and Gram-negative invasive pathogens.

Gentamicin is an aminoglycoside antibiotic with a wide spectrum of anti-bacterial activity, but however, due to its high solubility in water, it poorly penetrates inside the cells. This major inconvenient constitutes an important challenge for the treatment of intracellular bacterial infections, which might be solved using appropriate delivery systems for the targeted release of the bioactive agents at the intracellular sites of infection. Thus, in the case of antibiotics, the use drug delivery systems may contribute to increase their therapeutic activity against intracellular pathogens. This paper presents an efficient polymeric delivery system for the intracellular release of gentamicin based on bacterial polysaccharides.

Fluorescence analysis of apoptosis induced by Pseudomonas aeruginosa in endothelial cells.

Intracellular invasion of professional phagocytic cells like monocytes and macrophages by a pathogen usually triggers the apoptosis of the host cells. The aim of this study was to evaluate if Pseudomonas aeruginosa, although not considered a classic intracellular pathogen, could adhere to endothelial cell surface, invade the intracellular compartment and subsequently induce apoptosis of the cells. The adherence and invasion capacity of P. aeruginosa to endothelial cells was monitored using Cravioto's adapted method. The apoptotic cells were evidenced by staining with Acridine orange/Ethidium bromide. The qualitative assay of bacterial adherence to the cellular substrate revealed that all tested strains adhered to endothelial cells surface, exhibiting a diffuse, aggregative or mixed (diffuse-aggregative or localized-aggregative) pattern and 20-70% adherence rates. The adherence of P. aeruginosa induced the reorganization of cytoskeleton filaments and formation of endocytic membrane expansions. Cell free P. aeruginosa culture supernatants did not induce any cell death response, as noticed in case of whole bacterial culture, showing the capacity to induce apoptosis of endothelial cells. The fluorescence microscopy examination revealed chromatin condensation, fragmented nuclei, and membrane blebbing and apoptotic bodies in pathogen invaded cells.