Browsing by Author "Cunha, S."
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- Characterization of clinical and food Listeria monocytogenes isolates with different antibiotic resistance patterns through simulated gastrointestinal tract conditions and environmental stressesPublication . Cunha, S.; Komora, N.; Magalhães, R.; Almeida, Gonçalo; Ferreira, V.; Teixeira, PaulaThirty-three Listeria monocytogenes isolates previously collected from two sources, food (n = 18) and human patients suffering from listeriosis (n = 15), with variable antibiotic susceptibility profiles (sensitive/resistant) to erythromycin, ciprofloxacin and nitrofurantoin, were studied for their ability to survive (i) the environmental stress provided by sequential conditions that simulate the digestive tract, and (ii) extreme pH values (1.5–12). The results showed a response that was only strain dependent. There were no variability in survival results based on type of stress (low or high pH), source (food or clinical), or sensitivity/resistance to antibiotics (p > 0.01). Some strains of L. monocytogenes are able to survive extreme acid and alkaline conditions, and conditions that mimic the sequential stressors found in the gastro-intestinal tract. The resistance to the antibiotics tested in this study by some L. monocytogenes strains did not confer any cross-protection to acid or alkaline stressors.
- Effect of the application of bioactive extracts on the storage time of smoked horse mackerel (Trachurus trachurus) with reduced saltPublication . Barros, D.; Nova, P.; Cunha, S.; Barbosa, C.; Pintado, M.; Gomes, A.; Vaz-Velho, M.Smoking of food is one of the oldest preservation methods and quite popular among fish and meat preservation strategies. The process combines salting, drying, heating and smoking steps. Salting and smoking, besides their preserving effects, are important to add flavour so appreciated by traditional foods consumers1-3. However, reducing salt intake is an important public health issue. Thus, both food industry and food services are interested in reducing the salt content of products4. Moreover, today's consumer is also looking for more natural foods with high nutritional value and functionality 5,6. Special attention has been given to the addition of bioactive compounds mainly with antioxidant and antimicrobial activities7-9. The general objective of the present study was the valorisation of low commercial value and abundant fish species such as horse mackerel (Trachurus trachurus) by developing smoked products with reduced salt content and fortified with natural bioactive compounds extracted from seafood and forest by-products. The fish, obtained in the local auction market, was smoked in a semi-industrial smoking oven for 4 h at 70 °C with a final thermal shock step of 1 h at 90 °C. Smoked fillets were divided into four groups and sprayed with one of four bioactive extract solution (100 mg/ml): two different solutions of mussel extract with peptides <3kDa, one solution of microalgae extract, Tetraselmis sp. with peptides <3KDa and a pine bark extract (Pinus pinaster Aiton subsp. atlantica) solution. A control sample without any spraying completed the set of five samples. Smoked fillets were vacuum-packed and stored at 4-6 °C, over 30 d. Quality changes, over the 30 days of storage, were studied by monitoring microbiological and physicochemical properties at weekly intervals. For microbial enumeration a pack was opened weekly, and 30 g of smoked fish was taken aseptically, from different sites, and homogenised for 90 s in a stomacher and subsequently decimally diluted. Total viable counts were performed on pour plates according to EN ISO 4833-1:2013; psycrotrophic microorganisms according to ISO 17410:2001; Enterobacteriaceae counts according to ISO 21528-2:2017 and yeasts and molds according to NP 3277-1:1987. Detection of Salmonella and Listeria monocytogenes was done according to ISO 6579-1: 2017 and ISO 11290-1:2017, respectively. Physicochemical properties analysed included: salt (NaCl) content, aw, moisture, pH, peroxide value index (PV) and thiobarbituric acid index (TBA), antioxidant activity (DPPH and ABTS methods), colour and firmness. Sensory evaluation by quantitative descriptive analysis (11 attributes) was performed by four trained panellists. Average total viable cell numbers were similar among all samples (including control) except for mussel extract MuE (CPC) that maintained levels above 8 log CFU, form 2 wk of storage onwards; the remaining samples demonstrated a steady increase in total viable cell numbers (30 ºC) only from 21 d onwards and reached final numbers between 6-7 log CFU. Enterobacteriaceae were reported constantly between 1-2 log CFU, molds were undetectable and yeasts reached higher viable numbers (3 log CFU) in smoked fillets with mussel extracts. Salmonella and Listeria were not detected throughout storage. In terms of antioxidant activity samples were quite stable over time. Concerning degradation indicators, such as TBA and PV, no tendency was noticeable. TBA index after 7 d of storage was low and stable and PV was unstable. Nevertheless, panellists found some off-flavours during their evaluation especially in the samples with mussel extract and microalgae extract that was more intense after 15 d of storage (Figure 1). The pine bark-treated samples presented more similarity with the control samples throughout storage. Texture and firmness were generally stable over time and with small differences between treatments, control samples showing higher values. Principle component analysis applied to physicochemical data (Figure 2) showed that antioxidant activity and pH, moisture and water activity discriminate samples over time (PC1) and, sample treatment with mussel extract MuE(ESB) as well. In this output, samples with higher values are projected towards the left side and this was also clear in pine bark (PBE) treated samples. Samples with mussel extract (MuE(ESB)) are quite stable (PC1) except for those with 7 d of storage. Overall, these results indicate the potential of these treatments to extend shelf-life of fish products and contribute to reintroduce sea and forest by-products into the food chain along adding value to abundant and undervalued fish products.
- Immune responses and gut morphology of Senegalese sole (Solea senegalensis, Kaup 1858) fed monospecies and multispecies probioticsPublication . Batista, S.; Ramos, M. A.; Cunha, S.; Barros, Rui; Cristóvão, B.; Rema, P.; Pires, M. A.; Valente, L. M. P.; Ozório, R. O. A.The current study aimed to determine the effects of dietary probiotic supplementation on growth, gut morphology and non-specific immune parameters in Senegalese sole (Solea senegalensis) juveniles during a 1-month trial. Fish were fed for 1-month two diets with 1.0 or 4.6 × 106 CFU kg−1) of probiotic A (Bacillus sp., Pediococcus sp., Enterococcus sp. and Lactobacillus sp.) and two diets with 3.5 or 8.6 × 105 CFU kg−1 of probiotic B (Pediococcus acidilactici) and tested against an unsupplemented diet (control). Growth performance, as well as respiratory burst activity, nitric oxide (NO), alternative complement pathway (ACH50), lysozyme and peroxidase activities, was not affected by the dietary treatments. Probiotic supplementation tended to increased growth homogeneity between tanks having diet A1 the best possible alternative to decrease costs associated to size grading. Villous length and number of goblet cells of the anterior intestine did not vary among treatments. Muscle duodenal layer was significantly thicker in fish fed probiotic A compared to probiotic B, when included at the lowest level (A2 versus B2). The current study indicate that the use of the multispecies probiotic at 1.0 × 106 CFU kg−1 might enhance protection against pathogen outbreak and increase nutrient absorption, whereas at the highest concentration could reduced size dispersion among tanks.
- Molecular characterization of quinolone resistance mechanisms and extended-spectrum β-lactamase production in Escherichia coli isolated from dogsPublication . Meireles, D.; Leite-Martins, L.; Bessa, L. J.; Cunha, S.; Fernandes, R.; Matos, A. de; Manaia, C. M.; Costa, P. Martins daThe increasing prevalence of antimicrobial resistances is now a worldwide problem. Investigating the mechanisms by which pets harboring resistant strains may receive and/or transfer resistance determinants is essential to better understanding how owners and pets can interact safely. Here, we characterized the genetic determinants conferring resistance to β-lactams and quinolones in 38 multidrug-resistant Escherichia coli isolated from fecal samples of dogs, through PCR and sequencing. The most frequent geno-type included the β-lactamase groups TEM (n = 5), and both TEM + CTX-M-1 (n = 5). Within the CTX-Mgroup, we identified the genes CTX-M-32, CTX-M-1, CTX-M-15, CTX-M-55/79, CTX-M-14 and CTX-M-2/44. Thirty isolates resistant to ciprofloxacin presented two mutations in the gyrA gene and one or two mutations in the par C gene. A mutation in gyr A (reported here for the first time), due to a transversion andtransition (TCG → GTG) originating a substitution of a serine by a valine in position 83 was also detected.The plasmid-encoded quinolone resistance gene, qnrs1, was detected in three isolates. Dogs can be a reservoir of genetic determinants conferring antimicrobial resistance and thus may play an important role in the spread of antimicrobial resistance to humans and other co-habitant animals.
- Physiopathological responses of sole (Solea senegalensis) subjected to bacterial infection and handling stress after probiotic treatment with autochthonous bacteriaPublication . Peixoto, M.J.; Domingues, A.; Batista, S.; Gonçalves, J.F.M.; Gomes, A.M.; Cunha, S.; Ozorio, R.O.A.This study aimed to evaluate the protective effects of four autochthonous bacteria isolated from juvenile sole (Solea senegalensis) intestine as dietary probiotic supplement against bacterial pathogen infection and handling/transport stressors. Growth performance and immune responses were evaluated after 85 days of feeding trial. Sole (IBW=16.07 ± 0.11 g) were fed six experimental diets, a control diet (CTRL, without the dietary probiotic supplementation), and five diets supplemented with probiotic bacteria: PB1 (Shewanella hafniensis), PB2 (Enterococcus raffinosus), PB3 (Shewanella hafniensis + Arthrobacter soli), PB4 (Pseudomonas protegens + Arthrobacter soli) and PB5 (Shewanella hafniensis + Arthrobacter soli + Enterococcus raffinosus). All bacteria were selected based on their in vitro antimicrobial activity. After the growth trial, fish were submitted to a stress factor (transport) and then each dietary group was divided in two additional groups: non-infected (placebo) and infected with Photobacterium damselae subsp. piscicida. Immune and antioxidant responses were evaluated at day 10 post-infection. In infection trial A, fish were infected on the same day of transport, whereas in trial B fish were infected after a 7-day recovery from the transport stress. At the end of the feeding trial, fish fed with PB2 and PB4 showed lower final body weight when compared with the other dietary groups. Respiratory burst activity and nitric oxide production were not affected by probiotic supplementation. Fish fed with PB5 presented lower peroxidase activity compared to CTRL. Lysozyme and alternative complement pathway activity (ACH50) showed no significant differences between treatments. The innate immune responses were significantly affected after handling stress and bacterial infection. In trial A, the ACH50 levels of infected fish were significantly lower than the placebo groups. On the other hand, in trial B fish infected with Pdp demonstrated higher ACH50 levels when compared to placebos. Peroxidase levels were strongly modulated by bacterial infection and handling stress. In trials A and B, infection had a clear downgrade effect in peroxidase levels. Lipid peroxidation, catalase, glutathione S-transferase and glutathione reductase were altered by both bacterial infection and transport. Overall, dietary probiotic supplementation did not influence growth performance of sole. The immune and oxidative defenses of sole responded differently to infection depending on the probiotic and the synergy between pathogen infection and transport.
- Prevalence, Antimicrobial Susceptibility and Virulence Factors Profile of Methicillin- resistant Staphylococcus aureus (MRSA) on Food in PortugalPublication . Castro, A.; Palhau, C.; Cunha, S.; Camarinha, J.; Silva, Joana; Teixeira, Paula
- Virulence and resistance profile of Staphylococcus aureus isolated from foodPublication . Castro, A.; Palhau, C.; Cunha, S.; Camarinha, S.; Silva, J.; Teixeira, PStaphylococcus aureus is considered a global community and health care pathogen responsible for staphylococcal food poisoning. The aim of this study was to characterize several isolates of S. aureus recovered from different food products concerning enterotoxin genes and other virulence factors including antimicrobial resistance. In 2009, a total of 78 coagulase-positive staphylococci from 1454 food samples were identified to species level; 73 were confirmed as S. aureus. Of the S. aureus isolates 5.5% were resistant to oxacillin, 52.0% showed resistance to erythromycin, and 45.2% to tetracycline. Multidrug resistance was observed in 33.3% of the isolates (resistance to three or more antibiotics of different classes). SCCmec types IV and V were detected among methicillin-resistant S. aureus (MRSA). One MRSA isolate was pvl positive. The 52.0% of food isolates were shown to be enterotoxigenic; egc (63.0%), secbov (44.7%) were the main detected SEs. tst gene was also detected in food isolates. The present work demonstrates the presence of virulent S. aureus collected in 2009 in foods.