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- Rat olfactory mucosa mesenchymal stem/stromal cells (om-mscs): a characterization studyPublication . Alvites, Rui D.; Branquinho, Mariana V.; Caseiro, Ana R.; Amorim, Irina; Pedrosa, Sílvia Santos; Rêma, Alexandra; Faria, Fátima; Porto, Beatriz; Oliveira, Cláudia; Teixeira, Paula; Magalhães, Rui; Geuna, Stefano; Varejão, Artur S. P.; Maurício, Ana C.Stem/stromal cell-based therapies are a branch of regenerative medicine and stand as an attractive option to promote the repair of damaged or dysfunctional tissues and organs. Olfactory mucosa mesenchymal stem/stromal cells have been regarded as a promising tool in regenerative therapies because of their several favorable properties such as multipotency, high proliferation rate, helpful location, and few associated ethical issues. These cells are easily accessible in the nasal cavity of most mammals, including the rat, can be easily applied in autologous treatments, and do not cope with most of the obstacles associated with the use of other stem cells. Despite this, its application in preclinical trials and in both human and animal patients is still limited because of the small number of studies performed so far and to the nonexistence of a standard and unambiguous protocol for collection, isolation, and therapeutic application. In the present work a validation of a protocol for isolation, culture, expansion, freezing, and thawing of olfactory mucosa mesenchymal stem/stromal cells was performed, applied to the rat model, as well as a biological characterization of these cells. To investigate the therapeutic potential of OM-MSCs and their eventual safe application in preclinical trials, the main characteristics of OMSC stemness were addressed.
- Evaluation of antibiotic resistance patterns of food and clinical Listeria monocytogenes isolates in PortugalPublication . Barbosa, Joana; Magalhães, Rui; Santos, Isabel; Ferreira, Vânia; Brandão, Teresa R. S.; Silva, Joana; Almeida, Gonçalo; Teixeira, PaulaThe aim of this study was to characterize a broad collection of isolates of Listeria monocytogenes, of different serotypes, recovered in Portugal between 2003 and 2007 from foods (n = 353) and from clinical cases of human listeriosis (n = 95), in terms of antimicrobial susceptibility. All the isolates were susceptible to ampicillin, the preferred agent to treat listeriosis. Resistances to nitrofurantoin (n = 99), to ciprofloxacin (n = 18), to erythromycin (n = 10), to tetracycline (n = 2), to gentamicin (n = 1) and to rifampicin (n = 1) were observed. One hundred (28.3%) and 20 (21.0%) food and clinical isolates, respectively, were resistant to at least one antibiotic. Eight isolates (1.8%) were resistant to two or more antimicrobials of different classes, and all were collected from foods. Serogroup IVb included the highest percentage of isolates resistant to erythromycin. The highest percentages of isolates resistant to nitrofurantoin were of serogroup IVb and IIc. It was demonstrated that the incidence of antibiotic-resistant isolates of L. monocytogenes, during the period 2003 to 2007, was low in Portugal but still higher than that observed in other countries. Given the increasing population at greater risk of listeriosis, namely, the elderly, the high mortality rate of the infection and the detection of resistant isolates, monitoring for antibiotic resistance in strains of L. monocytogenes on a large scale, and assessing the risk of infection by these strains, is highly recommended.
- First confirmed human outbreak of Listeria monocytogenes in PortugalPublication . Almeida, G.; Magalhães, R.; Santos, I.; Ferreira, V.; Silva, J.; Mendes, M. M.; Nabais, P.; Fernandes, M. G. Mariano; Mâncio, M. I.; Sousa, M. M.; Teixeira, Paula
- Biofilm formation by persistent and non-persistent listeria monocytogenes strains on abiotic surfacesPublication . Magalhães, R.; Ferreira, V.; Biscottini, G.; Brandão, T. R. S.; Almeida, G.; Teixeira, PaulaContaminated food with Listeria monocytogenes is the predominant route of transmission of listeriosis to humans, a severe illness with a high mortality rate. Food processing environments can be colonized by persistent strains, repeatedly isolated for months or years. This study aimed to investigate the biofilm formation capacity of six strains of L. monocytogenes that persisted for long periods in two cheese processing plants in comparison to seven strains isolated sporadically from the same plants. Persistent strains produced more biofilm than non-persistent strains on stainless steel and silicon rubber surfaces; no significant differences were observed on polyvinyl chloride (PVC). In a polystyrene microtiter plate assay with crystal violet staining, no evidence was found that persistent strains have higher ability to form biofilm than non-persistent strains, and no correlation was identified between biofilm formation in the microtiter plate and on the three other surfaces tested.
- Study of Biological Hazards Present on the Surfaces of Selected Fruits and VegetablesPublication . Noronha, Lúcia; Castro, Ana; Ferreira, Vânia; Magalhães, Rui; Almeida, Gonçalo; Mena, Cristina; Silva, Joana; Teixeira, PaulaThis study evaluated the microbial load on the surface of fruits with rough and very pronounced textured peels, namely pineapples and melons (cantaloupe), and investigated the presence of foodborne pathogens in these products since they are usually eaten raw. Similarly, lettuce is one of the most common salad vegetables consumed raw in Portugal, it therefore being important to study the microbial status of lettuce leaves. Enumerations of aerobic mesophilic counts, Enterobacteriaceae, Escherichia coli and coagulase-positive staphylococci, as well as detection of Listeria monocytogenes and Salmonella spp., were performed for all samples. Only in melon samples were E. coli and coagulase-positive staphylococci not detected. Contamination with L. monocytogenes varied from 2.5% and 15% in pineapple/melon and lettuce samples, respectively. Salmonella spp. were not detected. All coagulase-positive staphylococci had similar characteristics and none exhibited multidrug resistance. L. monocytogenes isolates belonged to serogroups II (1/2c or 3c), or to serogroup IV (4b, 4d and 4e). None were found to be resistant to antibiotics commonly used in therapy of listeriosis.
- Impact of exposure to cold and cold-osmotic stresses on virulence-associated characteristics of Listeria monocytogenes strainsPublication . Alves, Ângela; Magalhães, Rui; Brandão, Teresa R. S.; Pimentel, Lígia; Rodríguez-Alcalá, Luis M.; Teixeira, Paula; Ferreira, VâniaThe objective of this work was to investigate the effect of stress conditions frequently encountered in food-associated environments on virulence-associated characteristics of eight strains of Listeria monocytogenes. Strains were grown at low (11 ºC, cold stress) and optimal (37 ºC) temperatures and in high NaCl concentrations (6% NaCl, 11 ºC; cold-osmotic stress) and tested for their ability to invade the human intestinal epithelial Caco-2 cells. Results demonstrate that the correlation between exposure to cold stress and increased invasion phenotype is strain-dependent as strains investigated exhibited different behaviours, i.e. exposure to cold stress conditions resulted in a significant increase of invasion levels in five out of the eight strains tested, when compared to growth under optimal conditions. On the other hand, when these cold-adapted cells were subsequently submitted to high salt concentrations and low temperature, their enhanced ability to invade Caco-2 was lost. Surprisingly, saturated fatty acids (SFA) and branched chain fatty acids (BCFA) decreased when L. monocytogenes were exposed to stress conditions as opposed to what has been observed in other studies, therefore highlighting that further studies will need to deepen in the understanding of the lipid metabolism of these strains. The effect of stress conditions on the survival of three selected L. monocytogenes strains through an in vitro gastrointestinal (GI) tract digestion model was further investigated. The exposure to cold-osmotic stress increased the survival of one strain through the GI tract.
- Detection of premature stop codons leading to truncated internalin A among food and clinical strains of Listeria monocytogenesPublication . Silva, Margarida Ferreira da; Ferreira, Vânia; Magalhães, Rui; Almeida, Gonçalo; Alves, Artur; Teixeira, PaulaListeria monocytogenes is a food-borne pathogen responsible for outbreaks and sporadic cases of listeriosis, a severe invasive disease. Internalin A (InlA) a protein encoded by inlA has a key role in the mechanism of pathogenesis in L. monocytogenes infection, specifically in the invasion of human intestinal epithelial cells. Studies on inlA have shown that mutations leading to premature stop codons (PMSCs) occur naturally and are associated with impaired virulence of L. monocytogenes strains. Increasing evidence suggests that inlA PMSCs mutations are frequent in strains from foods, but rare among clinical isolates. In this study, 22 L. monocytogenes strains collected in Portugal from the processing environment of a bakery industry (n = 1), different food products (n = 10) and human clinical cases (n = 11) were analysed for mutations in inlA and invasion efficiency in Caco-2 cells. Sequencing revealed previously reported mutations types leading to PMSCs in three food and one clinical strain presenting different molecular serotypes (i.e., IIa, IIb and IIc). The remaining 18 isolates did not show PMSCs in inlA. The four strains with PMSCs in inlA presented lower invasiveness efficiencies in Caco-2 cells (below 8.9%) when compared to the control strain (full-length InlA). In addition, one clinical isolate showed reduced invasion efficiency but no PMSCs in inlA. This isolate showed increased inlA transcript levels to that obtained for the laboratory control strain. Our data support the hypothesis that L. monocytogenes isolated from food have attenuated invasion due to the presence of inlA PMSCs. This information would be critically needed for adequate risk-assessments of the foodborne illness burden associated with L. monocytogenes strains.
- Study of Biological Hazards Present on the Surfaces of Selected Fruits and VegetablesPublication . Noronha, Lúcia; Magalhães, Rui; Silva, Joana; Teixeira, Paula
- Biocontrol strategies for Mediterranean-style fermented sausagesPublication . Oliveira, Marcia; Ferreira, Vânia; Magalhães, Rui; Teixeira, PaulaNaturally fermented meat sausages have a long tradition in Mediterranean countries and are one of the most important groups of traditional foods consumed throughout Europe. Despite all the advances in food science and technology and increased regulatory requirements and concerns for safety and quality during the last decades, the challenge to control important foodborne pathogens in this type of meat products still persists. Simultaneously, growing consumer interest in safe, high quality and minimal processed products, with less additives/preservatives have driven the food industry and scientists in a crusade for innovative technologies to maintain the safety of these products by natural means. Biological control (biocontrol) fits well within this tendency. This review summarizes the latest achievements on biocontrol strategies applied to Mediterranean-style fermented sausages, namely: (i) bioprotective cultures; (ii) bacteriocins; and, (iii) essential oils (EOs).
- Evaluation of a bacteriocin-producing strain of pediococcus acidilactici as a biopreservative for "alheira", a fermented meat sausagePublication . Albano, Helena; Pinho, Catarina; Leite, Daniela; Barbosa, Joana; Silva, Joana; Carneiro, Luísa; Magalhães, Rui; Hogg, Tim; Teixeira, PaulaThis study was conducted to evaluate the ability of Pediococcus acidilactici HA-6111-2, a PA-1 bacteriocinproducing lactic acid bacterium (LAB), isolated from ‘‘Alheira” to inhibit a cocktail of Listeria innocua strains during production and shelf-life of these products. The bacteriocinogenic culture reduced the Listeria population below the detection limit (1.5log CFU/g) and had no effect on the growth of the natural LAB flora or on the pH. Pathogenic organisms were not detected in any sample. The presence of some virulence factors and antibiotic resistances of the strain to be used as a bioprotective culture were investigated. P. acidilactici HA-6111-2 did not produce any of the biogenic amines tested; no formation of biofilms was observed; more L(+)lactic acid was produced than its isomer D(-); no gelatinase, DNase or lipase activity was recorded; no structural genes for the haemolysin, enterococcal surface protein, hydrolytic compounds, aggregation protein and cell-wall adhesins were detected, no significant antibiotic resistances were found. P. acidilactici HA-6111-2 appears to have potential as a bioprotective culture during ‘‘Alheira” fermentation. Moreover, a trained panel considered the protected product to be sensorially acceptable.
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