Lídia Dionísio

Occurrence of Salmonella spp in estuarine and coastal waters of Portugal.

The presence of Salmonella and its relationship with indicator organisms of fecal pollution, such as total coliforms, fecal coliforms and fecal streptococci, was studied at two marine zones in Portugal. Seventeen different Salmonella serotypes were isolated and identified, S. virchow was the most frequently isolated (21.6%). In addition, a high percentage (35.1%) was recorded for some Salmonella serotypes of clinical significance, namely S. enteritidis, S. infantis, S. typhimurium and S. virchow. In any of the samples from the two zones Salmonella was not detected in the absence of any of the indicator organisms. However, the incidence of Salmonella as a function of indicator concentration intervals established by the EEC standards was 0, 10 and 19.3% at guide values of total coliforms, fecal coliforms and fecal streptococci, respectively in the Faro samples (south of Portugal). In contrast, Salmonella incidence rates of 37.5, 36.4 and 33.3% were recorded at the corresponding guide values the Caminha samples (north of Portugal). No significant correlations (p>0.005) were obtained between Salmonella and the indicators at the sampling stations; however, total coliforms and fecal streptococci were the indicators most closely related to Salmonella in Caminha and Faro samples, respectively. Survival experiments in Escherichia coli, Enterococcus faecalis and S. typhimurium, using diffusion chambers, were performed to verify whether the lack of correlation between indicators and Salmonella was due to different inactivation rates in seawater. The results indicate that survival percentages of the three microorganisms tested were similar after 48 h of exposure to seawater.

The first detection of Leishmania major in naturally infected Sergentomyia minuta in Portugal

Phlebotomine sandflies of the genus Sergentomyia are widely distributed throughout the Old World. It has been suggested that Sergentomyia spp are involved in the transmission of Leishmania in India and Africa, whereas Phlebotomus spp are thought to be the sole vectors of Leishmania in the Old World. In this study, Leishmania major DNA was detected in one Sergentomyia minuta specimen that was collected in the southern region of Portugal. This study challenges the dogma that Leishmania is exclusively transmitted by species of the genus Phlebotomus in the Old World.

Microbiological Pollution of Ria Formosa (South of Portugal)

A 24-month survey on the microbiological quality of recreational shore marine waters in three sampling stations located at Ria Formosa (South of Portugal) was conducted. The classical indicators of faecal pollution, such as total coliforms (TC), faecal (thermotolerant) coliforms (FC), faecal streptococci (FS) and somatic coliphages, as well as several physico-chemical parameters (temperature, salinity, dissolved oxygen (DO) and transparency) were determined in the sampling stations, which exhibited different degrees of faecal pollution. The relationships between faecal indicators and several pathogenic microorganisms (Salmonella, Pseudomonas aeruginosa and Candida albicans) were also established in these recreational marine zones. The results obtained indicate that none of the indicators tested may be considered as a universal index of the presence of pathogens in water; however, faecal streptococci showed a higher and significant relationship with sewage discharges.

Leishmania infection and host-blood feeding preferences of phlebotomine sandflies and canine leishmaniasis in an endemic European area, the Algarve Region in Portugal

The Algarve Region (AR) in southern Portugal, which is an international tourist destination, has been considered an endemic region of zoonotic leishmaniasis caused by Leishmania infantum since the 1980s. In the present study, phlebotomine and canine surveys were conducted to identify sandfly blood meal sources and to update the occurrence of Leishmania infection in vectors and dogs. Four sandfly species were captured: Phlebotomus perniciosus, Phlebotomus ariasi, Phlebotomus sergenti and Sergentomyia minuta. In one P. perniciosus female, L. infantum DNA was detected. Blood meal tests showed that this species had no host preferences and was an opportunistic feeder. An overall canine leishmaniasis (CanL) seroprevalence of 16.06% was found; the seroprevalence was 3.88% in dogs housed in kennels and 40.63% in dogs that attended veterinary clinics. The simultaneous occurrence of dogs and P. perniciosus infected with L. infantum in the AR indicates that the region continues to be an endemic area for CanL. Our results reinforce the need for the systematic spatial distribution of phlebotomine populations and their Leishmania infection rates and the need to simultaneously perform pathogen monitoring in both invertebrate and vertebrate hosts to investigate the transmission, distribution and spreading of Leishmania infection.

Evaluation of media for the enumeration of faecal streptococci from natural water samples

The Most Probable Number technique using Azide dextrose broth and Ethyl violet azide dextrose broth, and the membrane filtration technique with the following agars: mEnterococcus, KF, Pfizer selective enterococcus, Kanamycin aesculin azide, Bile-Aesculin, Thallous acetate (Barne’s), and Mitis-Salivarius were compared on the basis of their accuracy, specificity, selectivity, precision, and relative recovery efficiency characteristics. The results obtained indicate that none of the above mentioned procedures shows an optimal selectivity. The Most Probable Number technique was a good procedure on the basis of its high recovery efficiency and good selectivity. However, mEnterococcus agar using the membrane filtration technique showed the best performance characteristics of the enumeration media tested, because this method possessed the best recovery efficiency, precision and accuracy, and also a good specificity.

Effect of packaging technology on microbiological and sensory quality of a cooked blood sausage, Morcela de Arroz, from Monchique region of Portugal.

Morcela de Arroz (MA), a popular Portuguese blood sausage, with high pH and water activity (aw), is traditionally commercialized without preservatives and unpacked. This study evaluated the best packaging solution to extend MA shelf life stored at 4±1°C for 44days: without packaging (WP), vacuum (VP) and modified atmosphere packaging (MAP) (80% CO2; 20% N2). Mesophilic (MTVC), psychrotrophic (PTVC), lactic acid bacteria (LAB), pseudomonads, molds and yeasts, Enterobacteriaceae, Listeria monocytogenes, Salmonella spp., Bacillus cereus, Clostridium perfringens, sensory properties, pH, moisture and aw were studied. Moisture and aw decreased (p<0.05) in WP. pH decreased in WP and MAP during storage. MTVC and PTVC counts increased to values around 7logCFU/g at 44days of storage. LAB and Enterobacteriaceae counts were higher (p<0.05) in VP. Pseudomonads were inhibited (p<0.05) by MAP after 8days of storage. Sensory parameters were affected (p<0.05) by packaging and storage time. Globally, MAP performed better.

Are the defined substrate-based methods adequate to determine the microbiological quality of natural recreational waters?

Monitoring the microbiological quality of water used for recreational activities is very important to human public health. Although the sanitary quality of recreational marine waters could be evaluated by standard methods, they are time-consuming and need confirmation. For these reasons, faster and more sensitive methods, such as the defined substrate-based technology, have been developed. In the present work, we have compared the standard method of membrane filtration using Tergitol-TTC agar for total coliforms and Escherichia coli, and Slanetz and Bartley agar for enterococci, and the IDEXX defined substrate technology for these faecal pollution indicators to determine the microbiological quality of natural recreational waters. ISO 17994:2004 standard was used to compare these methods. The IDEXX for total coliforms and E. coli, Colilert, showed higher values than those obtained by the standard method. Enterolert test, for the enumeration of enterococci, showed lower values when compared with the standard method. It may be concluded that more studies to evaluate the precision and accuracy of the rapid tests are required in order to apply them for routine monitoring of marine and freshwater recreational bathing areas. The main advantages of these methods are that they are more specific, feasible and simpler than the standard methodology.

Microbiological and Sanitary Quality of Recreational Seawaters of Southern Portugal

Three microbiological criteria (European Economic Community,World Health Organization/United Nations Environment Programand European Union) for water quality have been comparativelyevaluated in a resort zone of southern Portugal, located atthe Ria Formosa (Algarve). The results obtained in this studysuggest that the three compared directives adequatelyclassified the bathing zones according to their fecalpollution level. However, none of the directives establish asanitary classification of the recreational use of the bathingzones. By the use of the Cumulative Contamination Indexapplied in this study, classification based on both the levelof fecal pollution and on the sanitary condition of thebathing zones has been achieved. The application of this newapproach to the monitoring of bathing waters allows us topropose a new criterion of water quality based on thecoliphage levels (Ph80 = 300 pfu 100 mL-1 and Ph95 = 2000 pfu 100 mL-1) for inclusion in the newwater quality regulations. Coliphages have been shown to havea close correlation with the presence of pathogenic microorganisms, and with the fecal pollution levels of recreational seawaters. However, none of the microbial indicatorstested ensured detection of pathogen presence.

Simultaneous exposure of nematophagous fungi, entomopathogenic nematodes and entomopathogenic fungi can modulate belowground insect pest control

Entomopathogenic nematodes (EPNs) and fungi (EPF) are well known biological control agents (BCAs) against insect pests. Similarly, the nematophagous fungi (NF) are considered good BCA candidates for controlling plant parasitic nematodes. Because NF can employ EPNs as food and interact with EPF, we speculate that the simultaneous application of EPNs and EPF might result in higher insect mortality, whereas the triple species combination with NF will reduce the EPN and EPF activity by predation or inhibition. Here we evaluated single, dual (EPN + EPF, EPF + NF, EPN + NF) and triple (EPN + EPF + NF) combinations of one EPN, Steinernema feltiae (Rhabditida: Steinernematidae), one EPF, Beauveria bassiana (Hypocreales: Clavicipitaceae), and two NF, Arthrobotrys musiformis (Orbiliales: Orbiliaceae) and Purpureocillium lilacinum (Hypocreales: Ophiocordycipitaceae) under laboratory conditions. First, we showed that EPF reduced the growth rate of NF and vice versa when combined in both rich and limiting media, suggesting a negative interaction when combining both fungi. Three different fungal applications (contact with mycelia-conidia, immersion in conidial suspension, and injection of conidial suspension) were tested in single, dual and triple species combinations, evaluating Galleria mellonella (Lepidoptera: Pyralidae) larval mortality and time to kill. When mycelia was presented, the EPF appeared to be the dominant in combined treatments, whereas in immersion exposure was the EPN. In both types of exposure, NF alone did not produce any effect on larvae. However, when A. musiformis was injected, it produced larval mortalities >70% in the same time span as EPN. Overall, additive effects dominated the dual and triple combinations, with the exception of injection method, where synergisms occurred for both NF species combined with EPN + EPF. This study illustrates how differences in species combination and timing of fungal arrival can modulate the action of BCAs when augmented in the soil. Further studies are required to fine-tune these multitrophic interactions to provide successful, sustainable and resilient pest management in agroecosystems.

Report of in vitro antileishmanial properties of Iberian macroalgae

Abstract Here is reported the anti Leishmania infantum activity of 48 hexane, CH2Cl2 and MeOH extracts from 16 macroalgae collected on the Iberian Coast. Seven hexane and CH2Cl2 Cystoseira baccata, Cystoseira barbata, Cystoseira tamariscifolia, Cystoseira usneoides, Dictyota spiralis and Plocamium cartilagineum extracts were active towards promastigotes (IC50 29.8–101.8 μg/mL) inducing strong morphological alterations in the parasites. Hexane extracts of C. baccata and C. barbata were also active against intracellular amastigotes (IC50 5.1 and 6.8 μg/mL, respectively). Fatty acids, triacylglycerols, carotenoids, steroids and meroterpenoids were detected by nuclear magnetic resonance (NMR), and gas chromatography in the Cystoseira extracts. These results suggest that Cystoseira macroalgae contain compounds with antileishmanial activity, which could be explored as scaffolds to the development of novel sources of antiparasitic derivatives.