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- 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.
- 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.
- Behavior of Listeria monocytogenes isolates through gastro-intestinal tract passage simulation, before and after two sub-lethal stressesPublication . Barbosa, J.; Borges, S.; Magalhães, R.; Ferreira, V.; Santos, I.; Silva, J.; Almeida, G.; Gibbs, P.; Teixeira, P.The effects of previous exposure to sub-lethal acidic and osmotic stresses on the survival of Listeria monocytogenes during exposure to gastro-intestinal (GI) tract simulation, was investigated. Six L. monocytogenes strains isolated from cheeses were selected and exposed to high salt concentrations or acidic conditions and their viability compared in quick and slow digestions. The results demonstrated that (i) all isolates were more sensitive to the exposure to acidic than to osmotic sub-lethal conditions (ii) significant differences (p < 0.05) between the two types of digestion were observed; in slow digestion, the log reduction was higher for all the tested isolates (iii) all isolates were inhibited in the presence of bile salts for both types of digestion (iv) differences between quick and slow digestion were not observed (p > 0.05) after exposure to either osmotic or acidic stress (v) a higher cellular inactivation (p < 0.001) was observed during the passage through the GI tract simulation after exposure to osmotic than to acidic stresses and (vi) neither osmotic nor acidic sub-lethal stresses conferred resistance to simulated GI tract conditions.
- 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.
- Presence of microbial pathogens and genetic diversity of listeria monocytogenes in a constructed wetland systemPublication . Calheiros, Cristina S. C.; Ferreira, Vânia; Magalhães, Rui; Teixeira, Paula; Castro, PaulaThe potential of constructed wetlands (CWs) as a low technology for wastewater treatment is timely but there is a need to understand the route of pathogenic bacteria (Listeria monocytogenes and Salmonella spp.) and indicator organisms (Enterobacteriaceae and Escherichia coli) present in wastewater within those systems and to what extent they can compromise the further use of the plants (enabling in-house ornamental use) and water reuse (irrigation purposes). In the present study the fate of potential pathogenic bacteria within a CW, with ornamental plants, and an outlet pond established in a tourism house for wastewater treatment was assessed. The fate of pathogenic bacteria in lettuce, both plant tissues and soil, subject to irrigation with water contaminated either with L. monocytogenes or E. coli (indicator organism) was investigated. The uptake of indicator organisms by the CW ornamental plants was low and there was a decrease, from the CW wastewater inlet to the outlet, of up to 3 log for Enterobacteriaceae and up to 2 log for E. coli. Concerning Salmonella spp. no detection was registered in the water, plant tissues or substrate within the CW. On the other hand, several L. monocytogenes isolates were retrieved from water, root and substrate samples, found to have a high susceptibility to a battery of antibiotics. The irrigation of lettuce with water containing pathogenic bacteria (L. monocytogenes) and indicator organisms (E. coli) must be considered with caution since both bacteria were detected in the soil growing media and internalization of L. monocytogenes in roots was observed. These issues are poorly investigated and to our knowledge not addressed in the sphere of CWs.
- Foci of contamination of Listeria monocytogenes in different cheese processing plantsPublication . Almeida, G.; Magalhães, R.; Carneiro, L.; Santos, I.; Silva, J.; Ferreira, V.; Hogg, T.; Teixeira, P.Listeria monocytogenes is a ubiquitous bacterium widely distributed in the environment that can cause a severe disease in humans when contaminated foods are ingested. Cheese has been implicated in sporadic cases and in outbreaks of listeriosis worldwide. Environmental contamination, in several occasions by persistent strains, has been considered an important source of finished product contamination. The objectives of this research were to (i) evaluate the presence of L monocytogenes within the factory environments and cheeses of three processing plants, artisanal producer of raw ewe's milk cheeses (APC), small-scale industrial cheese producer (SSI) and industrial cheese producer (ICP) each producing a distinct style of cheese, all with history of contamination by L monocytogenes (ii) and identify possible sources of contamination using different typing methods (arsenic and cadmium susceptibility, geno-serotyping, PFGE). The presence of markers specific for 3 epidemic clones (ECI-ECIII) of L monocytogenes was also investigated: Samples were collected from raw milk (n = 179), whey (n = 3), cheese brining solution (n = 7), cheese brine sludge (n = 505), finished product (n = 3016), and environment (n = 2560) during, at least, a four-year period. Listeria monocytogenes was detected in environmental, raw milk and cheese samples, respectively, at 15.4%, 1.1% and 13.6% in APC; at 8.9%, 2.9% and 3.4% in SSI; and at 0%, 21.1% and 0.2% in ICP. Typing of isolates revealed that raw ewe's milk and the dairy plant environment are important sources of contamination, and that some strains persisted for at least four years in the environment. Although cheeses produced in the three plants investigated were never associated with any case or outbreak of listeriosis, some L monocytogenes belonging to specific PFGE types that caused disease (including putative epidemic clone strains isolated from final products) were found in this study.
- 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.
- Persistent and non-persistent strains of Listeria monocytogenes: A focus on growth kinetics under different temperature, salt, and pH conditions and their sensitivity to sanitizersPublication . Magalhães, Rui; Ferreira, Vânia; Brandão, Teresa R. S.; Casquete Palencia, R.; Almeida, Gonçalo; Teixeira, PaulaThis study aimed to investigate the effect of different conditions, including temperature (37 C, 22 C, and 4 C), NaCl concentrations (2.5%, 4%, and 8%), and acidity (pH ¼ 5), on the growth response of persistent and non-persistent isolates of Listeria monocytogenes. The resistance to two common sanitizers (benzalkonium chloride and hydrogen peroxide) was also investigated. A selected group of 41 persistent and non-persistent L. monocytogenes isolates recovered from three cheese processing plants during a previous longitudinal study was assembled. Average lag time was similar for persistent and non-persistent isolates grown at 37 C, 22 C and 4 C but significantly shorter (p < 0.05) for persistent isolates grown at 2.5%, 4% and 8% NaCl, and at pH 5. Average growth rates were significantly higher (p < 0.05) for persistent than for non-persistent isolates when grown at 22C, 2.5%, 4% and 8% NaCl, and at pH 5. These results suggest that persistent strains may be better adapted to grow under stressful conditions frequently encountered in food processing environments than non-persistent strains. No relation between persistence and resistance to the tested sanitizers was found.