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Peanut-based products have been associated with Salmonella foodborne outbreaks and/or recalls worldwide. The ability of Salmonella to persist for a long time in a low moisture environment can contribute to this kind of contamination. The objective of this study was to analyse the genome of five S. enterica enterica strains isolated from the peanut supply chain in Brazil, as well as to identify genetic determinants for survival under desiccation and validate these findings by phenotypic test of desiccation stress. The strains were in silico serotyped using the platform SeqSero2 as Miami (M2851), Javiana (M2973), Oranienburg (M2976), Muenster (M624), and Glostrup/Chomedey (M7864); with phylogenomic analysis support. Based on Multilocus Sequence Typing (MLST) the strains were assigned to STs 140, 1674, 321, 174, and 2519. In addition, eight pathogenicity islands were found in all the genomes using the SPIFinder 2.0 (SPI-1, SPI-2, SPI-3, SPI-5, SPI-9, SPI-13, SPI-14). The absence of a SPI-4 may indicate a loss of this island in the surveyed genomes. For the pangenomic analysis, 49 S. enterica genomes were input into the Roary pipeline. The majority of the stress related genes were considered as soft-core genes and were located on the chromosome. A desiccation stress phenotypic test was performed in trypticase soy broth (TSB) with four different water activity (aw) values. M2976 and M7864, both isolated from the peanut samples with the lowest aw, showed the highest OD570nm in TSB aw 0.964 and were statistically different (p < 0.05) from the strain isolated from the peanut sample with the highest aw (0.997). In conclusion, genome analyses have revealed signatures of desiccation adaptation in Salmonella strains, but phenotypic analyses suggested the environment influences the adaptive ability of Salmonella to overcome desiccation stress.

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The green crab (Carcinus maenas) is an inshore species affected by intertidal zonation patterns, facing periods of emersion during low tide and submersion during high tide. During these periods of air and subsequent water exposure, these species can face physiological challenges. We examined changes in O2 consumption rate (MO2), and ammonia and urea excretion rates over sequential 14 h periods in seawater (32 ppt, control), in air and during recovery in seawater after air exposure (13°C throughout). At the end of each exposure, the anterior (5th) and posterior (8th) gills and the hepatopancreas were removed for measurements of oxidative stress parameters (TBARs and catalase in the gills and hepatopancreas, and protein carbonyls in the gills). MO2 remained unchanged during air exposure, but increased greatly (3.4-fold above control levels) during the recovery period. Ammonia and urea net fluxes were reduced by 98% during air exposure, but rebounded during recovery to >2-fold the control rates. Exchangeable water pools, rate constants of diffusive water exchange, unidirectional diffusive water flux rates (using tritiated water) and transepithelial potential were also measured during control and recovery treatments, but exhibited no significant changes. Damage to proteins was not observed in either gill. However, lipid damage occurred in the anterior (respiratory) gill after the air exposure but not in the posterior (ionoregulatory) gill or hepatopancreas. Catalase activity also decreased significantly in recovery relative to levels during air exposure in both the anterior gill and hepatopancreas, but not in the posterior gill. The crabs did not modify water metabolism or permeability. We conclude that MO2 was maintained but not enhanced during air exposure, while ammonia and urea-N excretion were impaired. As a result, all of these parameters increase greatly during re-immersion recovery, and oxidative stress also occurs. Clearly, emersion is not without physiological costs.

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ABSTRACT: Salmonella is a relevant pathogen, which causes foodborne outbreaks associated with both high and low moisture foods (LMF). This study evaluated the effect of previous desiccation stress on the acid resistance of S. Typhimurium ATCC 14028 using blanched peanut kernels as an LMF model. Salmonella was recovered from the peanut samples throughout 180 days of blanched peanut kernels storage at 28 ºC. During this period two death rates were verified, 0.04 log cfu/g/day in the first 30 days and 0.007 log cfu/g/day between 30 and180 days.Regarding acid resistance, there was no difference (P > 0.05) in the Salmonella growth/death kinetics between the undesiccated sample (TSB)and the cells recovered from peanut samples over 180 days of storage after 4 h at pHs 3.0, 3.5, 4.5 and 7.2. The average growth rate observed for pH 7.2 was 0.44 log cfu/ml/h. At pH 4.5, the Salmonella counts did not change significantly over 4 h. In contrast, Salmonella populations declined by 0.14 to 0.29 log cfu/ml/h at pH 3.5. At pH 3.0 declines were estimated to be 0.65 log cfu/ml/h for the undesiccated sample and 2.07 log cfu/ml/h for Salmonella recovered from peanuts stored for 120 days. Therefore, our data indicated that desiccation stress caused during the peanut storagedid not influence the Salmonella acid resistance.

RESUMO: Em alimentos de baixa umidade (LMF), o primeiro desafio encontrado por patógenos como a Salmonella é o estresse de dessecação. Neste estudo, o efeito prévio do estresse de dessecação sobre a resistência ácida de S. Typhimurium ATCC 14028 foi avaliado utilizando amendoim blancheado como um modelo de LMF. Salmonella foi recuperada das amostras de amendoim após 180 dias de estocagem a 28 ºC. Durante este período foram verificadas duas taxas de mortalidade, 0,04 log ufc/g/dia nos primeiros 30 dias e 0,007 log ufc/g/dia entre 30 e 180 dias. Com relação à resistência ácida, não houve diferença (P > 0,05) na cinética de crescimento/morte de Salmonella entre a amostra sem estresse dessecativo (TSB) e as amostras de amendoim após 4 h em pHs 3,0, 3,5, 4,5 e 7,2. A taxa média de crescimento observada para o pH 7,2 foi de 0,44 log cfu/ml/h. No pH 4,5, a contagem de Salmonella não mudou significativamente durante 4 h. Em contraste, a população de Salmonella diminuiu de 0,14 a 0,29 log cfu/ml/h no pH 3,5. No pH 3,0, a queda foi estimada em 0,65 log cfu/ml/h para a amostra sem estresse dessecativo e 2,07 log cfu/ml/h para a Salmonella recuperada das amostras de amendoim estocadas por 120 dias. Portanto, nossos dados indicaram que o estresse dessecativo causado durante a estocagem do amendoim não influenciou a resistência ácida de Salmonella.

RESUMO

Salmonella is a relevant pathogen, which causes foodborne outbreaks associated with both high and low moisture foods (LMF). This study evaluated the effect of previous desiccation stress on the acid resistance of S. Typhimurium ATCC 14028 using blanched peanut kernels as an LMF model. Salmonella was recovered from the peanut samples throughout 180 days of blanched peanut kernels storage at 28 ºC. During this period two death rates were verified, 0.04 log cfu/g/day in the first 30 days and 0.007 log cfu/g/day between 30 and180 days.Regarding acid resistance, there was no difference (P > 0.05) in the Salmonella growth/death kinetics between the undesiccated sample (TSB)and the cells recovered from peanut samples over 180 days of storage after 4 h at pHs 3.0, 3.5, 4.5 and 7.2. The average growth rate observed for pH 7.2 was 0.44 log cfu/ml/h. At pH 4.5, the Salmonella counts did not change significantly over 4 h. In contrast, Salmonella populations declined by 0.14 to 0.29 log cfu/ml/h at pH 3.5. At pH 3.0 declines were estimated to be 0.65 log cfu/ml/h for the undesiccated sample and 2.07 log cfu/ml/h for Salmonella recovered from peanuts stored for 120 days. Therefore, our data indicated that desiccation stress caused during the peanut storagedid not influence the Salmonella acid resistance.

Em alimentos de baixa umidade (LMF), o primeiro desafio encontrado por patógenos como a Salmonella é o estresse de dessecação. Neste estudo, o efeito prévio do estresse de dessecação sobre a resistência ácida de S. Typhimurium ATCC 14028 foi avaliado utilizando amendoim blancheado como um modelo de LMF. Salmonella foi recuperada das amostras de amendoim após 180 dias de estocagem a 28 ºC. Durante este período foram verificadas duas taxas de mortalidade, 0,04 log ufc/g/dia nos primeiros 30 dias e 0,007 log ufc/g/dia entre 30 e 180 dias. Com relação à resistência ácida, não houve diferença (P > 0,05) na cinética de crescimento/morte de Salmonella entre a amostra sem estresse dessecativo (TSB) e as amostras de amendoim após 4 h em pHs 3,0, 3,5, 4,5 e 7,2. A taxa média de crescimento observada para o pH 7,2 foi de 0,44 log cfu/ml/h. No pH 4,5, a contagem de Salmonella não mudou significativamente durante 4 h. Em contraste, a população de Salmonella diminuiu de 0,14 a 0,29 log cfu/ml/h no pH 3,5. No pH 3,0, a queda foi estimada em 0,65 log cfu/ml/h para a amostra sem estresse dessecativo e 2,07 log cfu/ml/h para a Salmonella recuperada das amostras de amendoim estocadas por 120 dias. Portanto, nossos dados indicaram que o estresse dessecativo causado durante a estocagem do amendoim não influenciou a resistência ácida de Salmonella.

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One of the most important factors regulating the distribution and abundance of seaweeds is desiccation, triggered mainly by tidal changes and climatic variation. Porphyra and Pyropia species have evolved multiple strategies to tolerate desiccation stress; however, how these tolerance strategies differ in these species inhabiting different latitudes is still unknown. In this context, we analyzed, in situ, the physiological responses of these species (collected from 18° S to 41° S along the Chilean coast) to desiccation stress using biochemical and molecular analyses. The hyper-arid terrestrial climate of northern Chile, with high evaporation and lack of constant rain determines a very steep increase in desiccation stress in the upper intertidal during low tide for these species. Accordingly, the results showed that, in comparison with the southernmost populations, the Porphyra/Pyropia species from the north zone of Chile (18°-30° S) exhibited higher contents of lipoperoxide and carbonyls (1.6-1.9 fold) together with higher enzymatic activities, including ascorbate peroxidase, catalase, peroxiredoxin, and thioredoxin (2-3-fold). In addition, a substantial expression of cat, prx, and trx transcripts during desiccation was demonstrated, mainly in the northernmost populations. These results provide evidence of (i) significant activation of antioxidant enzymes and transcripts (principally cat and prx); (ii) participation of phenolic antioxidant compounds as a highly plastic physiological strategy to cope with desiccation; and (iii) the activation of the tolerance responses was affected by species latitudinal distribution. Thus, for the first time, this study integrated the biochemical and genetic responses of diverse Porphyra/Pyropia species to better understand their physiological dynamics of tolerance over a wide latitudinal range.

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In this study, we analyzed the antioxidant activity and total phenolic content of the intertidal seaweed Palisada perforata collected from different reef microhabitats (sheltered site, tide pool, plateau, and exposed site) along the coast of Pernambuco (Brazil). Both parameters were compared with the same parameters of this species grown in the laboratory under two experiments simulating temperature, salinity, and desiccation conditions found in the reef. After both experiments (temperature x salinity and desiccation), the algal photosynthetic performance was measured through chlorophyll fluorescence parameters using a pulse-amplitude modulation fluorometer to test their stress response. Palisada perforata likely underwent stress by desiccation due to tidal fluctuations rather than to temperature or salinity changes. This conclusion agrees with our observations of the plateau site´s specimens, which were exposed to both air and UV radiation during low tides and exhibited higher antioxidant activity to avoid oxidative damage. However, despite the environmental stress, the antioxidant activity remained low, suggesting that photoinhibition is a crucial protection mechanism against oxidative damage.

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MAIN CONCLUSION: The transcriptional modulation of desiccation tolerance factors in P. orbicularis explains its successful recuperation after water deficit. Differential responses to air exposure clarify seaweed distribution along intertidal rocky zones. Desiccation-tolerant seaweed species, such as Pyropia orbicularis, can tolerate near 96% water loss during air exposure. To understand the phenotypic plasticity of P. orbicularis to desiccation, several tolerance factors were assessed by RT-qPCR, Western-blot analysis, and enzymatic assays during the natural desiccation-rehydration cycle. Comparative enzymatic analyses were used to evidence differential responses between P. orbicularis and desiccation-sensitive species. The results showed that during desiccation, the relative mRNA levels of genes associated with basal metabolism [trehalose phosphate synthase (tps) and pyruvate dehydrogenase (pdh)] were overexpressed in P. orbicularis. Transcript levels related to antioxidant metabolism [peroxiredoxin (prx); thioredoxin (trx); catalase (cat); lipoxygenase (lox); ferredoxin (fnr); glutathione S-transferase (gst)], cellular detoxification [ABC transporter (abc) and ubiquitin (ubq)], and signal transduction [calmodulin (cam)] increased approximately 15- to 20-fold, with the majority returning to basal levels during the final hours of rehydration. In contrast, actin (act) and transcription factor 1 (tf1) transcripts were down-regulated. ABC transporter protein levels increased in P. orbicularis during desiccation, whereas PRX transcripts decreased. The antioxidant enzymes showed higher specific activity in P. orbicularis under desiccation, and sensitive species exhibited enzymatic inactivation and scarce ABC and PRX protein detection following prolonged desiccation. In conclusion, the reported findings contribute towards understanding the ecological distribution of intertidal seaweeds at the molecular and functional levels.

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The use of biochemical seed viability markers is often compromised by the unknown partitioning of analytes in bulk seed lots consisting of inseparable populations of viable and nonviable seeds. We took advantage of an unusual morphological syndrome found in the recalcitrant, undifferentiated seeds of Eugenia stipitata: one seed can be cut into several parts, each of which can germinate and develop into seedlings. We used four seed parts from one individual seed to analyse seed moisture content (MC), seed viability and the antioxidant glutathione (γ-glutamyl-cysteinyl-glycine; GSH), glutathione disulphide (GSSG) and intermediates of glutathione synthesis and breakdown. Seeds were exposed to different environmental MC to induce various levels of desiccation stress. Upon storage at high seed MC, seed viability was maintained, while GSH concentration increased and the glutathione half-cell reduction potential (EGSSG/2GSH ) was less negative than -215 mV, indicating GSH production and highly reducing conditions. Storage at low seed MC led to loss of GSH, resulting in a shift in EGSSG/2GSH , and seed death. In contrast, the cyst(e)ine half-cell reduction potential (ECySS/2CYS ) could not distinguish between the viability categories. Previous studies on seed populations revealed that the probability for a seed being alive is 50% at EGSSG/2GSH values between -180 and -160 mV. The single seed approach revealed that the window in which seed viability was lost could be slightly shifted towards more negative values. We discuss the contribution of cellular pH to EGSSG/2GSH and recommend E. stipitata as a recalcitrant seed model to study stress response on a single seed basis.

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