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- Bacteriophage Phi 6 as surrogate and human-harmless viruses to study anti-SARS-CoV-2 approachesPublication . Barros, Joana; Monteiro, Fernando; Ferraz, Maria PiaGiven safety challenges in conducting laboratory work with highly infectious human coronaviruses (pathogenicity, genetic mutations rate, biosafety level 3 and 4 requirements), many researchers have valued the potential of bacteriophages as appropriate viral surrogate to measure humans enveloped virus’ survival, transfer and removal. The use of phage Φ6 seems to be useful as coronavirus surrogate to assess the effectiveness of anti-SARS-CoV-2 approaches, providing important insights concerning COVID-19 pandemic and human public health.
- Detection of Staphylococcus aureus (MRSA/MSSA) in surfaces of dental medicine equipmentPublication . Gonçalves, Eva; Carvalhal, Rui; Mesquita, Rita; Azevedo, Joana; Coelho, Maria João; Magalhães, Ricardo; Ferraz, Maria Pia; Manso, M. Conceição; Gavinha, Sandra; Pina, Cristina Maria San Román Gomes de; Cardoso, Inês LopesMethicillin-Resistant Staphylococcus aureus (MRSA) represents one of the major causes of nosocomial infections, leading to high mortality. Surfaces in clinics, as well as the attending uniform and the hands of the dental doctor can be MRSA reservoirs. Having this in mind, the purpose of this study was to evaluate the presence of Methicillin-Sensitive Staphylococcus aureus (MSSA) and MRSA on dental medicine equipment surfaces. 354 Samples were collected from six equipment surfaces in six attendance areas before and after patient consultation and cultured in a selective medium. Polymerase Chain Reaction (PCR) was used to confirm the identity of bacterial strains as MRSA or MSSA. Data analysis was performed with chi-square tests with Bonferroni correction. It was observed 55.6% of uncontaminated samples. Contamination was: 17.5% MRSA (5.9% of samples collected before patient attendance and 11.6% after); 39.3% MSSA (14.1% collected before and 25.2% after). The prevalence of MRSA and MSSA was significantly higher after patient care. Integrated Clinic represented the most contaminated attendance area (MRSA 41.7%, MSSA 51.2%), the chair arm rest was the most contaminated surface for MRSA (29.7%) and the dental spittoon the most contaminated surface for MSSA (23.5%). Although a low level of contamination was observed, dental clinics, through patients possibly carrying bacteria, may be reservoirs for MRSA and MSSA transmission, and might contribute to potential nosocomial infections.
- Health monitoring and intervention plan on oil industry workers: results from a case-studyPublication . Oliveira, Gisela; Vidal, Diogo Guedes; Ferrari, Lilian; Costa, Andre; Ferraz, Maria PiaOil industry workers are particularly vulnerable to risks related to their especial working conditions like exposure to hazardous chemicals, explosions and fires, working in confined spaces and often in remote areas or in offshore platforms. Dedicated work health surveillance plans that take into consideration environmental risks are of the utmost importance to safeguard workers health and to communicate identified faults and gaps to other institutional departments to provide adequate intervention. This work presents an assessment case-study of an on-going health intervention and monitoring plan focused on oil industry workers and the quality of potable water distributed. In the assessment of risks to health, the quality of water for human use is case-sensitive as water is vital to life but may act as a transmission vector for several diseases whose symptoms may appear as acute (often as a consequence of water contamination by micro-organisms or toxic substances) or chronic, usually more related to the ingestion of chemically contaminated water. In the study timeline, six parameters were identified as critical in the water quality: Total Coliforms, Escherichia coli, iron, pH, turbidity and colour. A global graphical distribution of nonconformity anal-ysis by working service for each geographic location and for the entire period of the study highlighted the two worse water quality work sectors: ‘Oil Extractions Stations’ and ‘Baths & Changing Rooms’. Corrective measures arising from this case-study of the on-going health monitoring and intervention plan focus on sensibilization for improvement of workers hygiene and for cleaning and sanitization procedures.
- Staphylococcus aureusandEscherichia colidual-species biofilms on nanohydroxyapatite loaded with CHX or ZnO nanoparticlesPublication . Barros, Joana; Grenho, Liliana; Fontenente, Sílvia; Manuel, Cândida M.; Nunes, Olga C.; Melo, Luís F.; Monteiro, Fernando J.; Ferraz, Maria PiaImplant-associated infections are caused by surface-adhering microorganisms persisting as biofilms, resistant to host defense and antimicrobial agents. Given the limited efficacy of traditional antibiotics, novel strategies may rely on the prevention of such infections through the design of new biomaterials. In this work, two antimicrobial agents applied to nanohydroxyapatite materials-namely, chlorhexidine digluconate (CHX) and zinc oxide (ZnO) nanoparticles-were compared concerning their ability to avoid single- or dual-species biofilms of Staphylococcus aureus and Escherichia coli. The resulting biofilms were quantified by the enumeration of colony-forming units and examined by confocal microscopy using both Live/Dead staining and bacterial-specific fluorescent in situ hybridization. The sessile population arrangement was also observed by scanning electron microscopy. Both biomaterials showed to be effective in impairing bacterial adhesion and proliferation for either single- or dual-species biofilms. Furthermore, a competitive interaction was observed for dual-species biofilms wherein E. coli exhibited higher proliferative capacity than S. aureus, an inverse behavior from the one observed in single-species biofilms. Therefore, either nanoHA-CHX or nanoHA-ZnO surfaces appear as promising alternatives to antibiotics for the prevention of devices-related infections avoiding the critical risk of antibiotic-resistant strains emergence. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 491-497, 2017.
- Prevalence of antibiotic (ß-lactams, tetracycline, metronidazole, erythromycin) resistance genes in periodontic infectionsPublication . Freitas, Daniel; Gonçalves, Lara; Coelho, Maria João; Ferraz, Maria Pia; Magalhães, Ricardo; Pina, Cristina Maria San Román Gomes de; Cardoso, Inês LopesObjective: Porphyromonas gingivalis and Prevotella intermedia are thought to be pathogens in adult periodontitis. Antibiotherapy is usually needed in the treatment of periodontitis being often prescribed empirically. To allow prescription of a specific antibiotic treatment, identification of resistance genes should be performed. The aim of this study was the identification of the presence of TetM, TetQ, TEM, cfxA, MefA, ErmB and Nim resistance genes in previously identified P. intermedia and P. gingivalis isolated from samples collected from periodontal infections. Method: PCR was used for the identification of TetM, TetQ, TEM, cfxA, MefA, ErmB and Nim resistance genes in strains isolated from samples collected from periodontal infections. Results: It was seen that 8% of isolates had one of the tested tetracycline resistance genes. A total of 32% of β-lactamases resistance genes was observed in isolated strains. It was also observed that 2% of isolates had one of the analysed erythromycin resistance genes. None of the isolates showed the presence of the metronidazole resistance gene. Conclusions: Most strains harboring β-lactamase resistance genes had been previously identified as P. intermedia. No tetracycline resistance gene and a very low percentage of β-lactamase resistance genes were observed in P. gingivalis strains.
- Lytic bacteriophages against multidrug-resistant Staphylococcus aureus, Enterococcus faecalis and Escherichia coli isolates from orthopaedic implant-associated infectionsPublication . Barros, Joana; Melo, Luís D.R.; Poeta, Patrícia; Igrejas, Gilberto; Ferraz, Maria Pia; Azeredo, Joana; Monteiro, Fernando J.Orthopaedic implant-associated infections are a devastating complication of orthopaedic surgery with a significant impact on patients and healthcare systems. The aims of this work were to describe the patterns of antimicrobial resistance, pathogenicity and virulence of clinical bacterial isolates from orthopaedic implant-associated infections and to further isolate and characterise bacteriophages that are efficient in controlling these bacteria. Staphylococcus aureus, Enterococcus faecalis and Escherichia coli isolated from orthopaedic infections showed multiresistance patterns to the most frequently used antibiotics in clinical settings. The presence of mobile genetic elements (mecA, Tn916/Tn1545 and intl1) and virulence determinants (icaB, cna, hlb, cylLs, cylM, agg, gelE, fsr and fimA) highlighted the pathogenicity of these isolates. Moreover, the isolates belonged to clonal complexes associated with the acquisition of pathogenicity islands and antimicrobial resistance genes by recombination and horizontal gene transfer. Bacteriophages vB_SauM_LM12, vB_EfaS_LM99 and vB_EcoM_JB75 were characterised and their ability to infect clinical isolates of S. aureus, E. faecalis and E. coli, respectively, was assessed. Morphological and genomic analyses revealed that vB_EfaS_LM99 and vB_EcoM_JB75 belong to the Siphoviridae and Myoviridae families, respectively, and no genes associated with lysogeny were found. The bacteriophages showed low latent periods, high burst sizes, broad host ranges and tolerance to several environmental conditions. Moreover, they showed high efficiency and specificity to infect and reduce clinical bacteria, including methicillin-resistant S. aureus and vancomycin-resistant enterococci. Therefore, the results obtained suggest that the bacteriophages used in this work are a promising approach to control these pathogens involved in orthopaedic implant-associated infections.
- Efeitos da vancomicina, tetraciclina, tigeciclina e metronidazol na proliferação/viabilidade de uma linha de células osteoblásticasPublication . Pacheco, Ana Paula; Ferraz, Maria Pia; Coelho, Maria João; Alegrete, Nuno; Fernandes, Maria HelenaNeste estudo foram avaliados mecanismos citotóxicos de alguns antibacterianos usados nos tratamentos de infecções ósseas, nomeadamente vancomicina, tetraciclina, tigeciclina e metronidazol numa gama de concentrações de 0,005 μg/mL a 100 μg/mL. Relativamente à vancomicina não foi encontrada nenhuma evidência de citotoxicidade sobre as células MG63. A tetraciclina tem um efeito tóxico a partir da concentração de 0,5 μg/mL, efeito igualmente verificado na tigeciclina, no entanto para concentrações inferiores a 0,01 μg/mL parece ter ocorrido uma estimulação da divisão celular para este último antibiótico. No caso do metronidazol parece ocorrer logo uma diminuição da proliferação nas concentrações inferiores a 0,05 μg/mL. In this study the citoxicity of some antibacterials commonly used in bone infections, namely vancomycin, tetracycline, tigecyclin and metronidazol in a concentration range of 0.005 μg/mL to 100 μg/mL was evaluated. Vancomycin did not present any evidence of causing citotoxic effects to MG63 cells. Tetracycline did present toxic effects in concentrations above 0.5 μg/mL. The same phenomena was observed with tigecycline, however in low concentrations (below to 0.01 μg/mL) proliferation of cells seemed to be increased. In the case of metronidazol proliferation was diminished in concentrations below to 0.05 μg/mL.
- Anti-sessile bacterial and cytocompatibility properties of CHX-loaded nanohydroxyapatitePublication . Barros, J.; Grenho, Liliana; Fernandes, M.H.; Manuel, C.M.; Melo, L.F.; Nunes, O.C.; Monteiro, F.J.; Ferraz, Maria PiaNanohydroxyapatite possesses exceptional biocompatibility and bioactivity regarding bone cells and tissues, justifying its use as a coating material or as a bone substitute. Unfortunately, this feature may also encourage bacterial adhesion and biofilm formation. Surface functionalization with antimicrobials is a promising strategy to reduce the likelihood of bacterial infestation and colonization on medical devices. Chlorhexidine digluconate is a common and effective antimicrobial agent used for a wide range of medical applications. The purpose of this work was the development of a nanoHA biomaterial loaded with CHX to prevent surface bacterial accumulation and, simultaneously, with good cytocompatibility, for application in the medical field. CHX (5-1500 mg/L) was loaded onto nanoHA discs and the materials were evaluated for CHX adsorption and release profile, physic-chemical features, antibacterial activity against Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis, and cytocompatibility toward L929 fibroblasts. Results showed that the adsorption of CHX on nanoHA surface occurred by electrostatic interactions between the cationic group of CHX and the phosphate group of nanoHA. The release of CHX from CHX-loaded nanoHA showed a fast initial rate followed by a slower kinetics release, due to constraints caused by dilution and diffusion-limiting processes. NanoHA.50 to nanoHA.1500 showed strong anti-sessile activity, inhibiting bacterial adhesion and the biofilm formation. CHX-nanoHA caused a dose- and time-dependent inhibitory effect on the proliferation of fibroblasts for nanoHA.100 to nanoHA.1500. Cellular behavior on nanoHA.5 and nanoHA.50 was similar to control. Therefore, CHX-loaded nanoHA surfaces appear as a promising alternative to prevention of devices-related infections.
- Phase Behaviour and Miscibility Studies of Collagen/Silk Fibroin Macromolecular System in Dilute Solutions and Solid StatePublication . Ghaeli, Ima; de Moraes, Mariana; Beppu, Marisa; Lewandowska, Katarzyna; Sionkowska, Alina; Ferreira-da-Silva, Frederico; Ferraz, Maria Pia; Monteiro, FernandoMiscibility is an important issue in biopolymer blends for analysis of the behavior of polymer pairs through the detection of phase separation and improvement of the mechanical and physical properties of the blend. This study presents the formulation of a stable and one-phase mixture of collagen and regenerated silk fibroin (RSF), with the highest miscibility ratio between these two macromolecules, through inducing electrostatic interactions, using salt ions. For this aim, a ternary phase diagram was experimentally built for the mixtures, based on observations of phase behavior of blend solutions with various ratios. The miscibility behavior of the blend solutions in the miscible zones of the phase diagram was confirmed quantitatively by viscosimetric measurements. Assessing the effects of biopolymer mixing ratio and salt ions, before and after dialysis of blend solutions, revealed the importance of ion-specific interactions in the formation of coacervate-based materials containing collagen and RSF blends that can be used in pharmaceutical, drug delivery, and biomedical applications. Moreover, the conformational change of silk fibroin from random coil to beta sheet, in solution and in the final solid films, was detected by circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR), respectively. Scanning electron microscopy (SEM) exhibited alterations of surface morphology for the biocomposite films with different ratios. Surface contact angle measurement illustrated different hydrophobic properties for the blended film surfaces. Differential scanning calorimetry (DSC) showed that the formation of the beta sheet structure of silk fibroin enhances the thermal stability of the final blend films. Therefore, the novel method presented in this study resulted in the formation of biocomposite films whose physico-chemical properties can be tuned by silk fibroin conformational changes by applying different component mixing ratios.
- Development of silk fibroin/nanohydroxyapatite composite hydrogels for bone tissue engineeringPublication . Ribeiro, Marta; Moraes, Mariana A. de; Beppu, Marisa M.; Garcia, Mónica P.; Fernandes, Maria H.; Monteiro, Fernando J.; Ferraz, Maria PiaThis work presents a novel composite hydrogel consisting of silk fibroin (SF) and nanohydroxyapatite (nanoHA) prepared by a new and innovative method using ethanol as gelling agent capable of forming hydrogels in few minutes. The properties of the composite material, such as the microstructure as well as the chemical and physical properties were studied. Moreover in␣vitro studies of osteoblastic citocompatibility were performed. The microporosity and macroporosity obtained combined with interconnected porous structure and a uniform dispersion of nanoHA particles throughout the fibroin matrix makes composite hydrogel suitable for bone regeneration. The compression modulus of composite hydrogels was increased as the nanoHA concentration increased from 10 to 15 wt.% and the water uptake ability of these materials decreased with the incorporation of nanoHA. The metabolic and alkaline phosphatase activities of osteoblastic cells were improved with the incorporation of nanoHA in the SF matrix providing a more promising material for bone tissue engineering.