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- 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
- Characterisation of alheiras, traditional sausages produced in the North of Portugal, with respect to their microbiological safetyPublication . Ferreira, Vânia; Barbosa, Joana; Silva, Joana; Felício, Maria Teresa; Mena, Cristina; Hogg, Tim; Gibbs, Paul; Teixeira, PaulaThe objective of this study was the characterisation of alheiras, traditional Portuguese sausages, with respect to their microbiological safety. Thirty-eight lots from 17 producers were analysed. The microbiological status of the analysed product can be considered of concern in terms of food safety. Although Campylobacter spp. and Escherichia coli O157 were not detected in any sample,and Clostridium perfringens when present was not at levels of concern with reference to public health, Salmonella spp. were detected in 2 lots of industrially produced alheiras, and more than 60% of the lots analysed were contaminated with Listeria monocytogenes in concentrations higher than 100 cfu/g.
- Antilisterial active compound from lactic acid bacteria present on fresh iceberg lettucePublication . Ramos, Bárbara; Ferreira, Vânia; Brandão, Teresa R. S.; Teixeira, Paula; Silva, Cristina L. M.Pediococcus pentosaceus DT016, a bacteriocin producing strain, was isolated from fresh lettuce. A protein with antilisterial activity (bacDT016), between 11 to 17 kDa, was identifi ed and characterized as the bioactive substance from the LAB culture. Highest bacteriocin production was noted after 15 h of growth. Antibacterial activity was affected by some enzymes and detergents, as well as by temperatures equal or above 80 ºC. DT016 strain contains an 1110 bp DNA fragment with homology to pediocin AcH/PA-1.
- Cross-contamination events of Campylobacter spp. in domestic kitchens associated with consumer handling practices of raw poultryPublication . Cardoso, Maria João; Ferreira, Vânia; Truninger, Monica; Maia, Rui; Teixeira, PaulaContaminated poultry is the major vehicle for consumer's exposure to Campylobacter. This study aimed to perceive potential cross-contamination events during preparation of raw poultry that can contribute to the spread of Campylobacter spp. in domestic kitchen environments and to understand consumers' meanings and justifications on preparation of a poultry dish at home. A total of 18 households were visited to observe consumers preparing a recipe that included poultry. Poultry samples and swabs from the kitchen surfaces and utensils, such as kitchen cloth, hand towel, sponge, cutting boards and the sink, were collected before and after food preparation and tested for the presence of Campylobacter spp. Genotypic characterization of 72 Campylobacter spp. isolates was carried out through Pulse-Field Gel Electrophoresis (PFGE). Fourteen chicken samples were contaminated with Campylobacter spp. (77.8%). Twelve consumers (66.6%) washed the chicken meat under running tap water and eight (44.4%) used cutting boards. Also, only five consumers washed their hands properly prior to or during meal preparation. Cross-contamination events were detected in four kitchens, between the raw chicken and two cutting boards, two sinks and one kitchen cloth. The poultry samples presented different levels of contamination (< 4.0 × 101 CFU/g to 2.2 × 103 CFU/g), being some poultry with lower Campylobacter loads the origin of three cross-contamination events during food preparation. Both C. jejuni and C. coli were recovered. Molecular typing by PFGE showed a high diversity among the isolates. There were different explanations for the practice of cleaning and rinsing chicken, but, in general, it is an habit linked to what they have learned from their families. These results highlight the potential for the dissemination of Campylobacter strains in the domestic environment through the preparation of chicken meat and the need to raise awareness among consumers for an appropriate handling of raw poultry in order to decrease the risk of campylobacteriosis.
- 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.
- Effect of various protecting compounds added to the growth medium upon survival of lactobacillus sakei to heating, freezing, freeze-drying and storage in the dried statePublication . Ferreira, Vânia; Soares, Vânia; Santos, Cristina; Silva, Joana; Gibbs, Paul A.; Teixeira, PaulaThe aim of the present study was to investigate if the presence of sorbitol, myoinositol, xylose, mannose and Tween 80 in the growth medium increased the survival of L. sakei during heating, freezing, freeze drying and storage of freeze dried cells. Survival during freezing was enhanced by ca. 8% when glycerol was present in the growth medium. Viability during freeze-drying and storage in the dried state was improved by the presence of xylose in the growth medium. In addition to xylose, the presence of mannose and myoinositol protected cells during storage but not during drying. Cells grown in the presence myoinositol presented the highest D value at 55?C, 1.4 folder higher than control cells. In comparison with cells grown in MRS, total amino acids concentrations were the same order of magnitude for cells grown in the presence of mannose and myoinositol but were lower for cells grown in the presence of the other compounds.
- 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.