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- Microbiological and chemical quality of portuguese lettuce - results of a case studyPublication . Ferreira, Catarina; Lopes, Filipa; Costa, Reginaldo; Komora, Norton; Ferreira, Vânia; Fernandes, Virgínia Cruz; Delerue-Matos, Cristina; Teixeira, PaulaIn addition to environmental pollution issues, social concerns about the sustainability, safety, and quality of conventionally grown fruits and vegetables have been increasing. In order to evaluate if there were any microbiological differences between samples of organic and conventional lettuce, a wide range of parameters were tested, including pathogens and indicator organisms: the enumeration of Escherichia coli; the detection of Salmonella spp.; the detection/enumeration of Listeria monocytogenes; the enumeration of lactic acid bacteria, Pseudomonas spp. yeasts and molds, and Enterobacteriaceae. This study also evaluated the chemical safety of the lettuce samples, quantifying the nitrate concentration and 20 pesticides (14 organochlorine and 6 organophosphorus pesticides). Significant differences (p<0.05) between the conventional and organic samples were only detected for the counts of total microorganisms at 30◦C. Pathogens were absent in all the samples. The analytical method, using the quick, easy, cheap, effective, rugged, and safe (QuEChERS) approach for pesticide extraction, was suitable for detecting the targeted analyte s; the limit of quantification(LOQ) was between 0.6 and 1.8μg/kg (lower than the Maximum Residue Levels (MRLs) established by EU legislation). In three organic lettuce samples, one organochlorine pesticide (α-HCH) was observed below the MRLs. For the samples analyzed and for the parameters investigated, except for the total mesophilic counts, the organic and conventional lettuces were not different.
- 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.
- The protective effect of food matrices on Listeria lytic bacteriophage P100 application towards high pressure processingPublication . Komora, Norton; Bruschi, Carolina; Ferreira, Vânia; Maciel, Cláudia; Brandão, Teresa R. S.; Fernandes, Rui; Saraiva, Jorge A.; Castro, Sónia Marília; Teixeira, PaulaThe application of lytic phages as biocontrol agents is emerging as a promising strategy towards elimination or reduction of foodborne pathogens in a variety of food products. This technology is particularly advantageous for minimally processed and ready-to-eat (RTE) foods. In this study, the potential use of Listex™ P100 combined with high hydrostatic pressure (HPP), to enhance the control of Listeria monocytogenes in food was evaluated. For that, the effect of three pressures (200, 300 or 400 MPa; 5 min, 10 °C) on phage P100 stability was tested when inoculated in six different matrices: phosphate buffered saline (PBS, pH 7.4); apple juice (pH 3.41); orange/carrot nectar (pH 3.54); UHT whole milk (pH 6.73); and, two traditional Portuguese fermented products, “Serra da Estrela” cheese (pH 5.66) and “Alheira”, a meat sausage (pH 6.07). The results showed that treatment at 400 MPa reduced phage titres to below the detection level in all matrices, whereas at milder pressures the survival of the phage was matrix dependent. “Alheira”, “Serra da Estrela” cheese and UHT whole milk were shown to be baroprotective matrices that support phage P100 application in HHP up to 300 MPa; however, an accentuated phage inactivation was observed in apple and orange/carrot nectar, which may be related to the acidic pH values of these matrices. The initial phage load did not affect the inactivation rate during HHP processing (300 MPa, 5 min, 10 °C) in PBS, cheese, sausage or milk matrices, and the phage titres were stable in these matrices during storage at 4 °C for 28 days for milk and 60 days for “Alheira” and “Serra da Estrela” cheese. In addition, a baroprotective effect on phage stability was observed when PBS was supplemented with reducing sugars, dextrin, casein, and tween 80. In conclusion, at mild HHP treatment, phage P100 remained active in specific matrices and seems to present potential to be added in non-thermal inactivation of L. monocytogenes.
- Prevalence of staphylococcus aureus from nares and hands on health care professionals in a portuguese hospitalPublication . Castro, A.; Komora, N.; Ferreira, Vânia; Lira, A.; Mota, M.; Silva, Joana; Teixeira, PaulaAims: The main goal was to estimate the prevalence of methicillin-resistant Staphylococcus aureus on hands and in nose of health care professionals. Methods and Results: Detection of Staph. aureus on hands or in the nose of 169 individuals was performed. Nasal and hand carriage was found in 39 6 and in 8 9% respectively. About 17 2% of the individuals were carriers of methicillin-resistant Staph. aureus (MRSA) in the nose and 4 7% on hands. The majority of nasal MRSA were resistant to b-lactams, erythromycin and ciprofloxacin. All nasal MRSA were SCCmec type IV and Panton-Valentine leukocidin (PVL) negative. One MRSA isolated from hand was SCCmec type V. About 75 6% of MRSA isolates presented the same or closely related restriction patterns. Sixty per cent of Staph. aureus from hands and from noses from the same individual were the same strain. Conclusions: MRSA nasal carriage was high considering healthy health care professionals but in accordance with high level of MRSA infection in Portugal. Isolates recovered in this study seemed to be different from major clones previously isolated in other Portuguese hospitals. Significance and Impact of the Study: These findings may have implications on the knowledge of healthy health care workers as vehicles of MRSA infections among the community. Presence of several virulence factors may contribute to increased pathogenesis in case of infection.
- Non-thermal approach to Listeria monocytogenes inactivation in milk: the combined effect of high pressure, pediocin PA-1 and bacteriophage P100Publication . Komora, Norton; Maciel, Cláudia; Pinto, Carlos A.; Ferreira, Vânia; Brandão, Teresa R. S.; Saraiva, Jorge M. A.; Castro, Sónia Marilia; Teixeira, PaulaNon-thermal food processing and replacement of chemical additives by natural antimicrobials are promising trends in the food industry. The objective of the present work was to evaluate the effect of a process which combines mild high hydrostatic pressure – HHP (200 and 300 MPa, 5 min, 10 °C), phage Listex™ P100 and the bacteriocin pediocin PA-1 as a new non-thermal process for destruction of Listeria monocytogenes (104 CFU mL−1 or 107 CFU mL−1) in milk. For inoculum levels of 104 CFU mL−1, HHP combined with phage P100 eliminated L. monocytogenes immediately after pressurization. When L. monocytogenes was inoculated at levels of 107 CFU mL−1, a synergistic effect between phage P100, pediocin PA-1 and HHP (300 MPa) on the inactivation of L. monocytogenes was observed during storage of milk at 4 °C. For non-pressure treated samples inoculated with phage or pediocin or both, L. monocytogenes counts decreased immediately after biocontrol application, but regrowth was observed in a few samples during storage. Phage particles were stable during refrigerated storage for seven days while pediocin PA-1 remained stable only during three days. Further studies will have to be performed to validate the findings of this work in specific applications (e.g. production of raw milk cheese).
- Survival of listeria monocytogenes with different antibiotic resistance patterns to food-associated stressesPublication . Komora, Norton; Bruschi, Carolina; Magalhães, Rui; Ferreira, Vânia; Teixeira, PaulaThe ongoing rise of antibiotic resistant microbial pathogens has become one of the major public health threats worldwide. Despite all the effort and actions taken so far, a proliferation of antibiotic resistant (AR) and multi-antibiotic resistant (MAR) strains is still observed, including in foodborne pathogens. This trend has been also noted recently for isolates of Listeria monocytogenes, a species that, although remaining largely sensitive to clinically relevant antimicrobials, has been reported to develop increased tolerance to antibiotics, particularly in isolates recovered from the food-chain. In this study we compared the ability of MAR (n=8), AR (n=18) and antibiotic susceptible (AS, n=11) L. monocytogenes strains from food and clinical origin to survive to different environmental stress conditions, including temperature (58°C), acidic stress (1% v/v lactic acid, pH3.5), and osmotic stress (37% w/v NaCl). The presence of antibiotic active efflux among MAR and AR strains, and its role on L. monocytogenes tolerance to different antimicrobial compounds was also investigated, namely; hydrogen peroxide; organic acids (acetic, citric and lactic); nisin; benzalkonium chloride (BC); and, sodium nitrite. While no significant differences were observed in the survival of the 37 strains exposed to high temperature (58°C), overall the mean logarithmic reduction of clinical strains was statistically lower after acid and salt exposure than that observed for strains of food origin; but both food and clinical strains resistant to two or three antibiotics were significantly less susceptible to acid (lactic acid 1% v/v) and osmotic stresses (37% w/v NaCl) when compared to AS strains. Using the EtBr-agar Cartwheel method, it was possible to detect efflux pumps in three of the 26 MAR and AR isolates, including one control strain; the active efflux in theses isolates was proven to be associated with fluoroquinolone resistance, and possible extrusion of BC and hydrogen peroxide. The mechanisms responsible for the possible correlation between resistance to antibiotics and to acid or salt stress in L. monocytogenes have yet to be understood.
- High hydrostatic pressure effects on Listeria monocytogenes and L. innocua: evidence for variability in inactivation behaviour and in resistance to pediocin bacHA-6111-2Publication . Bruschi, Carolina; Komora, Norton; Castro, Sónia Marília; Saraiva, Jorge; Ferreira, Vânia Borges; Teixeira, PaulaThe effect of high hydrostatic pressure (HHP) on the survival of 14 strains of Listeria monocytogenes from food or clinical origins, selected to represent different pheno and genotypes, was evaluated. Stationary phase cells were submitted to 300, 400 and 500 MPa at 10 °C, for 5 min. A high variability in the resistance of L. monocytogenes to pressure was observed, and particularly two strains isolated from food were significantly more baroresistant than the rest. Strains of L. monocytogenes resistant to one or more antibiotics exhibited significantly higher levels of survival after the high pressure treatment at 400 MPa. No correlation was found between strains' origin or thermal tolerance and resistance to HHP. The suitability of two strains of L. innocua as surrogates of L. monocytogenes, was also investigated. These exhibited significantly higher sensitivities to HHP than observed for some L. monocytogenes. The antimicrobial effect of the antilisterial bacteriocin (bacHA-6111-2) increased after L. monocytogenes cells had been exposed to pressure. The data obtained underlines the importance of strain selection for studies aiming to evaluate HHP efficacy to ensure safety of HHP-treated foods.