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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
- Listeriosis and Listeria monocytogenes in Portugal : from surveillance studies to persistence in food processing plantsPublication . Magalhães, Rui Miguel Barros de Sousa; Teixeira, Paula Cristina Maia; Almeida, Gonçalo António Nieto Uria Ribeiro deListeria monocytogenes is a foodborne pathogen capable of causing severe human disease (listeriosis) when contaminated foods are ingested, particularly in groups at higher risk for listeriosis: the very young, old, immunocompromised individuals and pregnant women. Its ubiquity and ability to adapt and survive under extreme conditions (e.g., refrigeration temperatures, wide pH range, high salt concentrations), makes this pathogen of difficult eradication and control in the food-processing environment. Contaminated readyto- eat foods that support the growth of the pathogen are a major concern. Crosscontamination by the equipment and the general food processing environment is one of the most important sources of food contamination. Some strains may persist in food processing environment over several months/years, while others are only sporadically recovered. Although it is general accepted that particular traits of these strains contribute to their persistence in food processing environment, specific characteristics of strains that confer better survival/adaptation to the food processing environment remain unclear but are essential for planning preventive measures. Previous studies performed in the CBQF (Centro de Biotecnologia e Química Fina – Centre of Biotechnology and Fine Chemistry) demonstrated that strains that caused human listeriosis were isolated from cheeses and the cheese processing plants. The overall goal of this work was to integrate applied research and outreach to augment knowledge and try to define control strategies regarding L. monocytogenes persistence in food processing environment and monitoring the cases of listeriosis in Portugal. Information regarding cases of listeriosis, and when available the isolate that caused the disease, have been collected from the main hospitals in Portugal between 2008 and 2012. A total of 203 cases of listeriosis were detected. The annual incidence rate observed ranged from 0.2 to 0.7 cases per 100,000 inhabitants. The mean age of the nonmaternal/neonatal (non-MN) cases with documented age was 59 years, and 46.4% occurred in patients aged over 65 years. Clinical isolates were characterized by genoserotyping, resistance to arsenic and cadmium and DNA macrorestriction analysis by pulsed field gel electrophoresis. The minimal inhibitory concentrations of antimicrobials was also determined. Several clusters of isolates presenting different geographic and time distributions were detected. The incidence of antibiotic-resistant isolates of L. monocytogenes was low but significantly higher than in previous years (2003-2007). This study, involving 25 national hospitals, led to the detection of an outbreak that occurred between March 2009 and February 2012. Of the 30 cases of listeriosis reported, 27 were in the Lisbon and Vale do Tejo region. The case fatality rate was 36.7%. All cases were caused by molecular serogroup IVb isolates indistinguishable by pulsed-field gel electrophoresis and ribotype profiles. Collaborative investigations with the national health and food safety authorities identified cheese as the probable source of infection, traced to a processing plant. A previous longitudinal study carried by our research team identified persistence in from two cheese processing plants. A selected group of 41 persistent and non-persistent L. monocytogenes isolates was assembled for this study. The effect of different conditions, including temperature (37 ºC, 22 ºC, and 4 ºC), NaCl concentrations (2.5%, 4%, and 8%), and acidity (pH = 5), on the growth response of persistent and non-persistent isolates of L. monocytogenes was determine; the resistance to two common sanitizers (benzalkonium chloride and hydrogen peroxide) was also investigated. Results suggest that persistent strains may be more adapted to grow under stressful conditions frequently encountered in food processing environments, like 22 ºC, 2.5%, 4% and 8% NaCl, and at pH 5, than nonpersistent strains. No relation between persistence and resistance to the tested sanitizers was found. For the group of 41 isolates a new selection of a six persistent and seven nonpersistent strains isolated from the same processing plants were evaluated for biofilm formation in stainless steel, silicon rubber, and polyvinyl chloride (PVC) coupons; a microplate titer assay was also carried out. Persistent strains produced more biofilm than non-persistent strains in stainless steel and silicon rubber surfaces; but no significant differences were observed in PVC. In the polystyrene microtiter plate assay stained with cristal violet 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 in the three other surfaces tested. The continuous subtyping of isolates is essential and the study of persistence of L. monocytogenes in food processing plants is important to develop new and more efficient strategies for control of this pathogen.
- Clinical isolates of L. monocytogenes: serotyping, biotyping and susceptibility to antibioticsPublication . Almeida, G.; Ferreira, V.; Magalhães, R.; Barbosa, J.; Silva, J.; Teixeira, P.; Hogg, T.