Compositional analysis of biofilms formed by Staphylococcus aureus isolated from food sources

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2016.00390

Characterization of microbial populations in a wastewater treatment plant focusing on Staphylococcus aureus and Pseudomonas aeruginosa

Dissertação de mestrado em Bioengenharia

Evaluation of antibacterial activity of caffeic acid encapsulated by -cyclodextrins

Context: Caffeic acid is described as antibacterial, but this bioactive molecule has some issues regarding solubility and stability to environmental stress. Thus, encapsulation devices are required. Objective: The aim of this work was to study the effect of the caffeic acid encapsulation by cyclodextrins on its antibacterial activity. Materials and methods: The interactions between the caffeic acid and three cyclodextrins (-cyclodextrin (CD), 2-hydroxypropyl--cyclodextrin (HPCD) and methyl--cyclodextrin were study. Results and discussion: The formation of an aqueous soluble inclusion complex was confirmed for CD and HPCD with a 1:1 stoichiometry. The CD/caffeic acid complex showed higher stability than HPCD/caffeic acid. Caffeic acid antibacterial activity was similar at pH 3 and pH 5 against the three bacteria (K. pneumoniae, S. epidermidis and S. aureus). Conclusions: The antibacterial activity of the inclusion complexes was described here for the first time and it was shown that the caffeic acid activity was remarkably enhanced by the cyclodextrins encapsulation.

Antibacterial properties of fibrous stents coated with silver oxide nanocoatings

Stents are rigid and perforated tubular structures, which are inserted into blood vessels in order to prevent or inhibit the constriction of blood flow, restoring the normal blood flow, when blood vessels are clogged, being used in 70% of angioplasties. These medical devices assume great importance in the treatment of cardiovascular diseases (CVD) which are the leading cause of death worldwide. In the European Union CVD account for 40% of deaths and assume an estimated annual cost of 196 billion euros[1]. Stents must possess certain requirements, in order to, adequately, perform its function, such as biocompatibility (so that its use does not c ause damage on the health of its user), mechanical strength, radiopacity (so that it is easy to view), longitudinal flexibility, ease of handling, corrosion resistance and having high strength and high radial expansion ability to recover. Stents can be made of different materials, but metals, particularly stainless steel, are the most common. However, metallic stents present several dRawbacks such as corrosion and restenosis, leading to health complications for the patient, or even death. In order to minimize these disadvantages, new materials, like fibrous materials, have been used [2]. Monofilaments present high potential for stents development because, in addition to its biocompatibility, these materials allow the application of various surface treatments, such as antibacterial coatings. Furthermore, monofilament exhibit excellent mechanical properties, like greater stiffness and good results when subjected to compression, tensile and bending forces, since these forces will be directly supported by the monofilament [3]. To minimize the reaction of the human body and Limit the adhesion of microorganisms to the stent surface, some coatings have been developed, including the use of novel metals with antimicrobial properties, like silver. The main objective of this study was the development of fibrous stents, incorporation of silver oxide nanocoating. For the development of the stent, polyester monofilaments with 0.27mm of diameter were used in braiding technology, with a mandrel diameter of 6mm and a braiding angle of 35⁰. The mechanical behaviour of the stent were evaluated by mechanical testing under longitudinal and radial compression, bending. The results of compressive strength tests are according with value from literature: 1.13 to 2.9 N for radial compression and 0. 16-5.28N to longitudinal compression. From literature is also possible to verify that stents must present 75% of unchanged diameter during the bending test and must possess a porosity between 70% and 80% [4]. The produced polyester stent presents values of 1.29N for radial compression, 0.23N for longitudinal compression, 80% of porosity and 85.5% of unchanged diameter, during bending tests. For the antibacterial functionalization, silver oxide nanocoatings were prepared, through reactive magnetron g, with an Ag target in an Ar +O2 atmosphere. In order to evaluate the nanostructure and morphology of the coatings, d ifferent technique s like X-ray diffraction (XRD), scanning electron microscopy (SEM) and and X- ray photoelectron spectroscopy (XPS were used. From the analyses of XRD it is possible to verify that the peaks corresponds to planes of Ag2 O and MATERIAIS 2015 Porto, 21-23 June, 2015 characterize a cubic phase. The presence of Ag2 O is corroborated by XPS spectrum, where it is possible to observe silver, not only, in oxide state, but a lso in mettalic state, and it is possible to verify the presence of silver clusters, confirmed by SEM analysis. Films’ roughness and topography, parameters influencing the wettability of the surface and microorganism adhesion, were measured by Atomic Force Microscopy (AFM), and it was observed that the roughness is very low (under 10 nm). Coatings’ hydrophobicity and surface tension parameters were determined by contact angle measurement, and it was verified the hydrophobic behavior of the coatings. For antibacterial tests were used Staphylococcus epidermidis strain (IE186) and Staphylococcus aureus(ATCC 6538), and halo inhibition zone tests were realized. Ag+release rates were studied by means of inductively coupled plasma mass spectrometry (ICP -MS). The obtained results suggest that silver oxide coatings do not modify significantly surface properties of the substrate, like hydrophobicity and roughness, and present antimicrobial properties for both bacteria used.

Comparing different methods to measure biofilm thickness: the techniques are: optical coherence tomography, confocal laser scanning microscopy and low load compression test

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

PEGylated ofloxacin nanoparticles render strong antibacterial activity against many clinically important human pathogens.

The rise of bacterial resistance against important drugs threatens their clinical utility. Fluoroquinones, one of the most important classes of contemporary antibiotics has also reported to suffer bacterial resistance. Since the general mechanism of bacterial resistance against fluoroquinone antibiotics (e.g. ofloxacin) consists of target mutations resulting in reduced membrane permeability and increased efflux by the bacteria, strategies that could increase bacterial uptake and reduce efflux of the drug would provide effective treatment. In the present study, we have compared the efficiencies of ofloxacin delivered in the form of free drug (OFX) and as nanoparticles on bacterial uptake and antibacterial activity. Although both poly(lactic-co-glycolic acid) (OFX-PLGA) and methoxy poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) (OFX-mPEG-PLGA) nanoformulations presented improved bacterial uptake and antibacterial activity against all the tested human bacterial pathogens, namely, Escherichia coli, Proteus vulgaris, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus, OFX-mPEG-PLGA showed significantly higher bacterial uptake and antibacterial activity compared to OFX-PLGA. We have also found that mPEG-PLGA nanoencapsulation could significantly inhibit Bacillus subtilis resistance development against OFX.

Incorporation of antimicrobial peptides on functionalized cotton gauzes for medical applications

A large group of low molecular weight natural compounds that exhibit antimicrobial activity has been isolated from animals and plants during the past two decades. Among them, peptides are the most widespread resulting in a new generation of antimicrobial agents with higher specific activity. In the present study we have developed a new strategy to obtain antimicrobial wound-dressings based on the incorporation of antimicrobial peptides into polyelectrolyte multilayer films built by the alternate deposition of polycation (chitosan) and polyanion (alginic acid sodium salt) over cotton gauzes. Energy dispersive X ray microanalysis technique was used to determine if antimicrobial peptides penetrated within the films. FTIR analysis was performed to assess the chemical linkages, and antimicrobial assays were performed with two strains: Staphylococcus aureus (Gram-positive bacterium) and Klebsiella pneumonia (Gram-negative bacterium). Results showed that all antimicrobial peptides used in this work have provided a higher antimicrobial effect (in the range of 4 log–6 log reduction) for both microorganisms, in comparison with the controls, and are non-cytotoxic to normal human dermal fibroblasts at the concentrations tested.

Morphology and oxygen incorporation effect on antimicrobial activity of silver thin films

Ag and AgxO thin films were deposited by non-reactive and reactive pulsed DC magnetron sputtering, respectively, with the final propose of functionalizing the SS316L substrate with antibacterial properties. The coatings were characterized chemically, physically and structurally. The coatings nanostructure was assessed by X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS), while the coatings morphology was determined by scanning electron microscopy (SEM). The XRD and XPS analyses suggested that Ag thin film is composed by metallic Ag, which crystallizes in fcc-Ag phase, while the AgxO thin film showed both metallic Ag and Ag-O bonds, which crystalize in fcc-Ag and silver oxide phases. The SEM results revealed that Ag thin film formed a continuous layer, while AgxO layer was composed of islands with hundreds of nanometers surrounded by small nanoparticles with tens of nanometers. The surface wettability and surface tension parameters were determined by contact angle measurements, being found that Ag and AgxO surfaces showed very similar behavior, with all the surfaces showing a hydrophobic character. In order to verify the antibacterial behavior of the coatings, halo inhibition zone tests were realized for Staphylococcus epidermidis and Staphylococcus aureus. Ag coatings did not show antibacterial behavior, contrarily to AgxO coating, which presented antibacterial properties against the studied bacteria. The presence of silver oxide phase along with the development of different morphology were pointed as the main factors in the origin of the antibacterial effect found in AgxO thin film. The present study demonstrated that AgxO coating presented antibacterial behavior and its application in cardiovascular stents is promising.

Zeolite-based nanomaterials for the control of opportunistic pathogenic microorganisms

Dissertação de mestrado em Applied Biochemistry (área de especialização em Biomedicine)

Bacterial cellulose-lactoferrin as an antimicrobial edible packaging

Bacterial cellulose (BC) films from two distinct sources (obtained by static culture with Gluconacetobacter xylinus ATCC 53582 (BC1) and from a commercial source (BC2)) were modified by bovine lactoferrin (bLF) adsorption. The functionalized films (BC+bLF) were assessed as edible antimicrobial packaging, for use in direct contact with highly perishable foods, specifically fresh sausage as a model of meat products. BC+bLF films and sausage casings were characterized regarding their water vapour permeability (WVP), mechanical properties, and bactericidal efficiency against two food pathogens, Escherichia coli and Staphylococcus aureus. Considering their edibility, an in vitro gastrointestinal tract model was used to study the changes occurring in the BC films during passage through the gastrointestinal tract. Moreover, the cytotoxicity of the BC films against 3T3 mouse embryo fibroblasts was evaluated. BC1 and BC2 showed equivalent density, WVP and maximum tensile strength. The percentage of bactericidal efficiency of BC1 and BC2 with adsorbed bLF (BC1+bLF and BC2+bLF, respectively) in the standalone films and in inoculated fresh sausages, was similar against E. coli (mean reduction 69 % in the films per se versus 94 % in the sausages) and S. aureus (mean reduction 97 % in the films per se versus 36 % in the case sausages). Moreover, the BC1+bLF and BC2+bLF films significantly hindered the specific growth rate of both bacteria. Finally, no relevant cytotoxicity against 3T3 fibroblasts was found for the films before and after the simulated digestion. BC films with adsorbed bLF may constitute an approach in the development of bio-based edible antimicrobial packaging systems.

Influence of oxygen conditions on bacterial interactions within biofilms related with cystic fibrosis

Dissertação de mestrado em Bioengineering