Acute chest syndrome in children with sickle cell disease: Data from a national AIEOP cohort identify priority areas of intervention in a hub-and-spoke system.

Acute chest syndrome (ACS) is a frequent cause of hospitalization in sickle cell disease (SCD). Despite advances in acute care, many settings still lack knowledge about ACS best practices. After the AIEOP Guidelines were published in 2012, suggesting standardized management in Italy, a retrospective study was performed to assess the diagnostic and therapeutic pathways of ACS in children. From 2013 to 2018, 208 ACS episodes were presented by 122/583 kids in 11 centres. 73 were male, mean age 10.9 years, 85% African, 92% HbSS or Sß°. In our hub-and-spoke system, a good adherence to Guidelines was documented, but discrepancies between reference centres and general hospitals were noted. Improvement is needed for timely transfer to reference centres, use of incentive spirometry, oxygen therapy and pain management.

Oxylipins generation in Lactobacillus helveticus in relation to unsaturated fatty acid supplementation.

AIMS: Oxylipins are regarded as unsaturated fatty acids (UFAs) oxidation products, whose accumulation in plants and fungi is associated with stress. The aim of this study was to investigate if a metabolic pathway from UFAs to oxylipins was present also in lactic acid bacteria (LAB). METHODS AND RESULTS: A strain of Lactobacillus helveticus, incubated in the presence of oleic, linoleic and linolenic acids released, after 2 h, fatty acid oxidation products, mainly C6 , C8 , C9 aldehydes and alcohols. An experiment with total carbon labelled linoleic acid, in the presence or not of an oxidative stress, demonstrated that oxylipins, such as hexanal, octanal, nonanal, 2-octenal, 2-octanal, originated mainly from the oxidation of this fatty acid. CONCLUSIONS: Since lipoxygenase, dioxygenase and cytochrome P450 genes have never been found in L. helveticus, a possible pathway for linoleic conversion and oxylipins formation could include, as a first step, the transient formation of hydroxylated linoleic acids by fatty acids hydratases. However, the sequence of steps from the linoleic acid to the C6 and C8 aldehydes needs to be more deeply investigated. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the multiple role of oxylipins which are flavouring agents, antimicrobial compounds and interspecific signalling molecules, the knowledge of the mechanisms involved in their biosynthesis in food related bacteria could have an important biotechnological impact, also allowing the overproduction of selected bioactive molecules.

High pressure homogenization versus heat treatment: effect on survival, growth, and metabolism of dairy Leuconostoc strains.

The effect of high pressure homogenization (HPH) with respect to a traditional heat treatment on the inactivation, growth at 8°C after treatments, and volatile profile of adventitious Leuconostoc strains isolated from Cremoso Argentino spoiled cheeses and ingredients used for their manufacture was evaluated. Most Leuconostoc strains revealed elevated resistance to HPH (eight passes, 100 MPa), especially when resuspended in skim milk. Heat treatment was more efficient than HPH in inactivating Leuconostoc cells at the three initial levels tested. The levels of alcohols and sulfur compounds increased during incubation at 8°C in HPH-treated samples, while the highest amounts of aldehydes and ketones characterized were in heated samples. Leuconostoc cells resuspended in skim milk and subjected to one single-pass HPH treatment using an industrial-scale machine showed remarkable reductions in viable cell counts only when 300 and 400 MPa were applied. However, the cell counts of treated samples rose rapidly after only 5 days of storage at 8°C. The Leuconostoc strains tested in this work were highly resistant to the inactivation treatments applied. Neither HPH nor heat treatment assured their total destruction, even though they were more sensitive to the thermal treatment. To enhance the inhibitory effect on Leuconostoc cells, HPH should be combined with a mild heat treatment, which in addition to efficient microbial inactivation, could allow maximal retention of the physicochemical properties of the product.

Depuration of striped venus clam (Chamelea gallina L.): effects on microorganisms, sand content, and mortality.

This study was focused on the evaluation of the microbiological indices, defined by European legislation, before and after the depuration of clams (Chamelea gallina) landed in category B seawater. The survival of depurated clams and the meat yield were also evaluated. The results obtained from October 2002 to September 2003 evidenced a mean microbial reduction during depuration of 62% for Escherichia coli and 54% for fecal coliforms (FC). All the samples had FC counts below the limit after 24 h depuration with the exception of the August samples. E. coli was found in concentration slightly higher than the legal limit only in the samples of December and January. In August, the E. coli concentration did not decrease during the depuration, while in the other samples significant reduction of E. coli concentration was observed. Salmonella spp. and V. parahaemolyticus were never detected in the clams harvested between March and September. Vibrio alginolyticus was found in the clams harvested in May and September both before and after the depuration process. The viability of clam was not negatively affected by depuration, in fact, an increase of viability was observed with the exception of the samples of April. The meat yield was not influenced by the depurative treatment in C. gallina; the mean value found before depuration, 10.47% (with 1.95 SD), did not significantly vary after the treatment (10.58%, SD 2.07). In conclusion, the depuration conditions can improve the quality of C. gallina; however, its effects on microbiological quality depended on environmental conditions.

Effect of capric, lauric and alpha-linolenic acids on the division time distributions of single cells of Staphylococcus aureus.

The effect of non-inhibitory concentrations of capric, lauric and alpha-linolenic acids (C10:0, C12:0 and C18:3 respectively) on the division time distribution of single cells of Staphylococcus aureus was evaluated at pH 7 and pH 5. The effect of the initial cell concentration on the lag time of growing cell populations was also assessed. The statistical properties of the division times (defined as the time interval from birth to next binary fission for a single cell) were studied using the method of Elfwing et al. [Elfwing, A., Le Marc, Y., Baranyi, J., Ballagi, A., 2004. Observing the growth and division of large number of individual bacteria using image analysis. Applied and Environmental Microbiology 70, 675-678]. The division times were significantly longer in the presence of free fatty acids than in the control. Shorter division intervals were detected at pH 7 than at pH 5 in the control experiment and in the presence of C10:0. However, both C12:0 and C18:3 slowed down the growth, regardless of the pH. The observed division time distributions were used to simulate growth curves from different inoculum sizes using the stochastic birth process described by Pin and Baranyi [Pin, C., Baranyi, J., 2006. Kinetics of single cells: observation and modelling of a stochastic process. Applied and Environmental Microbiology 72, 2163-2169]. The output of the simulation results were compared with observed data. The lag times fitted to simulated growth curves were in good agreement with those fitted to growth curves measured by plate counts. The averaged out effect of the population masked the effect of the free fatty acids and pH on the division times of single cells.

Growth and metabolites production by Penicillium brevicompactum in yoghurt.

Penicillium brevicompactum, commonly encountered in the indoor air, is known to produce a mycotoxin, mycophenolic acid (MPA). This mould has been isolated from a wide range of foods; considering that we had previously isolated this species from contaminated yoghurt, in this study we have evaluated its growth in yoghurt sweetened with sucrose, fructose and fructose added with fruit pieces. Fungal growth was evaluated monitoring CO(2) production in the headspace during yoghurt storage at 4+/-1, 8+/-1 and 10+/-1 degrees C throughout 21 days. P. brevicompactum grew well in the samples sweetened with fructose at 8 and 10 degrees C. The addition of sucrose influenced the growth negatively, particularly at 4 degrees C. Volatile Organic Compounds (VOC) and MPA production was determined at 8 degrees C in inoculated and uninoculated yoghurt, as well as in liquid malt extract. Differences in VOC profiles and in MPA production were correlated with the age of the fungus and with the growth medium. This study points out for the first time the early qualitative changes in volatile production patterns of a common indoor mould, grown in yoghurt, as well as the production of MPA during storage at refrigeration temperatures.

Probiotic Crescenza cheese containing Lactobacillus casei and Lactobacillus acidophilus manufactured with high-pressure homogenized milk.

High-pressure homogenization (HPH) is one of the most promising alternatives to traditional thermal treatment of food preservation and diversification. Its effectiveness on the deactivation of pathogenic and spoilage microorganisms in model systems and real food is well documented. To evaluate the potential of milk treated by HPH for the production of Crescenza cheese with commercial probiotic lactobacilli added, 4 types of cheeses were made: HPH (from HPH-treated milk), P (from pasteurized milk), HPH-P (HPH-treated milk plus probiotics), and P-P (pasteurized milk plus probiotics) cheeses. A strain of Streptococcus thermophilus was used as starter culture for cheese production. Compositional, microbiological, physicochemical, and organoleptic analyses were carried out at 1, 5, 8, and 12 d of refrigerated storage (4 degrees C). According to results obtained, no significant differences among the 4 cheese types were observed for gross composition (protein, fat, moisture) and pH. Differently, the HPH treatment of milk increased the cheese yield about 1% and positively affected the viability during the refrigerated storage of the probiotic bacteria. In fact, after 12 d of storage, the Lactobacillus paracasei A13 cell loads were 8 log cfu/ g, whereas Lactobacillus acidophilus H5 exhibited, in P-P cheese, a cell load decrease of about 1 log cfu/g with respect to the HPH-P cheese. The hyperbaric treatment had a significant positive effect on free fatty acids release and cheese proteolysis. Also, probiotic cultures affected proteolytic and lipolytic cheese patterns. No significant differences were found for the sensory descriptors salty and creamy among HPH and P cheeses as well as for acid, piquant, sweet, milky, salty, creamy, and overall acceptance among HPH, HPH-P, and P-P Crescenza cheeses.

Effect of high-pressure homogenization, nonfat milk solids, and milkfat on the technological performance of a functional strain for the production of probiotic fermented milks.

The aim of this research was the evaluation of the effects of milkfat content, nonfat milk solids content, and high-pressure homogenization on 1) fermentation rates of the probiotic strain Lactobacillus paracasei BFE 5264 inoculated in milk; 2) viability loss of this strain during refrigerated storage; and 3) texture parameters, volatile compounds, and sensorial properties of the coagula obtained. The data achieved suggested a very strong effect of the independent variables on the measured attributes of fermented milks. In fact, the coagulation times were significantly affected by pressure and added milkfat, and the rheological parameters of the fermented milk increased with the pressure applied to the milk for added nonfat milk solids concentrations lower than 3%. Moreover, the polynomial models and the relative response surfaces obtained permitted us to identify the levels of the 3 independent variables that minimized the viability loss of the probiotic strain used during refrigerated storage.

Generation of aroma compounds in sourdough: effects of stress exposure and lactobacilli-yeasts interactions.

The effects of the interaction between Saccharomyces cerevisiae LBS and Lactobacillus sanfranciscensis LSCE1 and of their responses to acid, oxidative or osmotic stress on alcohol and aroma production were assessed. The exposure of S. cerevisiae LBS and L. sanfranciscensis LSCE1 cells to oxidative, acid or osmotic sub-lethal stress gave rise to a common or specific responses. Gamma-decalactone, 2(5H)-furanones and aldehydes were overproduced by LAB following oxidative stress. The acid stress induced both in yeasts and LAB, as well as in their co-cultures, a relevant accumulation of isovaleric and acetic acids and higher alcohols. A cross-exposure of yeasts and LAB to their preconditioned media, generated in S. cerevisiae a release of esters including esters of long-chain unsaturated fatty acids coming from membrane phospholipids. These esters were excreted also by yeasts following a pressure stress.

Characterization of yeasts involved in the ripening of Pecorino Crotonese cheese.

The aims of this work were to identify and characterize for some important technological properties the yeast species present throughout the ripening process of Pecorino Crotonese, a traditional cheese produced in a well defined area of Southern Italy. In particular, the strain technological properties considered include fermentation/assimilation of galactose and lactose, assimilation of lactate and citrate in the presence of different NaCl concentrations, hydrolysis of butter fat, skim milk, gelatine and casein, production of brown pigments in cheese agar and ability to produce biogenic amines. High yeast levels were recorded in cheese samples already after 5 h of brining (about 5 log cfu/g) and these concentration remained constant during ripening. The yeast isolates belonged to restrict number of yeast species. While Kluyveromyces lactis and Saccharomyces cerevisiae were isolated prevalently in the first stages of Pecorino Crotonese production, Yarrowia lipolytica and Debaryomyces hansenii dominated during the later stages of maturation. Otherwise, the latter two were very NaCl resistant species. In fact, D. hansenii strains conserved the ability to assimilate lactose and galactose in the presence of 10% NaCl, while almost all the strains of Y. lipolytica isolated assimilated citrate and lactate up to 7.5% NaCl. Y. lipolytica isolates evidenced also the highest proteolytic and lipolytic activities and the capability to catabolize tyrosine producing brown pigment. In addition they resulted in the highest aminobiogenic potential decarboxylating ornithine, phenylalanine, tyrosine and lysine. However, they were not able to produce histamine, biogenic amine produced by three strains of D. hansenii.

Use of Yarrowia lipolytica strains for the treatment of olive mill wastewater.

The principal aim of this research was to evaluate the ability of different Yarrowia lipolytica strains, having different origin, to grow in olive mill wastewater (OMW) and reduce its COD level. All the strains were able to grow in undiluted OMW; the comparison between the data obtained in a semi-synthetic medium and in OMW suggests that lipases with different specificity can be produced in relation to the medium composition. Under the adopted conditions, the reduction of the OMW COD values varied from 1.47% and 41.22% of the initial value. Some strains determined a significant reduction of polyphenol content, while other ones caused its apparent increase. Moreover, some Y. lipolytica strains, isolated from chilled foods, produced the highest citric acid concentrations. These results evidenced that some Y. lipolytica strains are good candidates for the reduction of the pollution potential of OMW and for the production of enzymes and metabolites such as lipase and citric acid.

Interactions between high pressure homogenization and antimicrobial activity of lysozyme and lactoperoxidase.

It was the objective of this work to evaluate the effect of high pressure homogenization on the activity of antimicrobial enzymes such as lysozyme and lactoperoxidase against a selected group of Gram positive and Gram negative species inoculated in skim milk. Lactobacillus helveticus, Lactobacillus plantarum and Listeria monocytogenes were the most pressure resistant species while Bacillus subtilis, Pseudomonas putida, Salmonella typhimurium, Staphylococcus aureus, Proteus vulgaris and Salmonella enteritidis were found to be very sensitive to the hyperbaric treatment. The enzyme addition enhanced the instantaneous pressure efficacy on almost all the considered species as indicated by their instantaneous viability loss following the treatment. Moreover, the combination of the enzyme and high pressure homogenization significantly affected the recovery and growth dynamics of several of the considered species. Although L. monocytogenes was slightly sensitive to pressure, the combination of the two stress factors induced a significant viability loss within 3 h and an extension of lag phases in skim milk during incubation at 37 degrees C. The hypothesis formulated in this work is that the interaction of high pressure homogenization and lysozyme or lactoperoxidase is associated to conformational modifications of the two proteins with a consequent enhancement of their activity. This hypothesis is supported by the experimental results also regarding the increased antimicrobial activity against L. plantarum of the previously pressurised lysozyme with respect to that of the native enzyme.

Automated ribotyping and random amplified polymorphic DNA analysis for molecular typing of Salmonella enteritidis and Salmonella typhimurium strains isolated in Italy.

AIMS: The ability of automated ribotyping and random amplified polymorphic DNA (RAPD) analysis to differentiate Salmonella enteritidis and Salmonella typhimurium isolates in relation to their origin was evaluated. METHODS AND RESULTS: The restriction enzymes EcoRI, PvuII and PstI, and the random primers OPB17 and P1254, were tested for ribotyping and RAPD analysis, respectively. Seventeen subtypes were identified among the isolates of the two pathogenic Salmonella serovars using the RiboPrinter, and 25 subtypes using RAPD. CONCLUSIONS: The greatest degree of genetic diversity was observed among Salm. typhimurium isolates using both automated ribotyping (Simpson's index of discrimination 0878) and RAPD (Simpson's index of discrimination 0886). SIGNIFICANCE AND IMPACT OF THE STUDY: According to the results of this research, automated ribotyping and RAPD are two useful genotyping techniques for identifying unique and common subtypes associated with a specific source and location, and provide powerful tools for epidemiological investigations.

Proteolytic, lipolytic and molecular characterisation of Yarrowia lipolytica isolated from cheese.

This work studied the qualitative and quantitative proteolytic and lipolytic activities of Yarrowia lipolytica strains isolated from two cheese types. Randomly amplified polymorphic DNA-PCR (RAPD-PCR) analysis was used to compare the cheese strains of Y. lipolytica with strains isolated from other food products and with the type strain of the species in order to investigate the genetic diversity and occurrence of specific environmental groups. Diversity of proteolytic and especially lipolytic activity within Y. lipolytica strains isolated from dairy products was observed. In particular, the degree of specificity for saturated or unsaturated fatty acids as well as for even- or odd-numbered carbon free fatty acids (FFAs) varied among the strains. The RAPD-PCR profiles showed low genetic relatedness between many of the food isolates and the type strain of the species. Such genetic variability needs to be further evaluated. Most of the Y. lipolytica strains appeared to be specific to the particular environment from which they were isolated. However, phenotypic characteristics having technological importance in dairy products and, particularly, lipolytic activities did not correspond to the genetic differences observed by RAPD-PCR analysis.

Variability of the lipolytic activity in Yarrowia lipolytica and its dependence on environmental conditions.

This work was aimed to the evaluation of the variability of lipolytic activity in Yarrowia lipolytica strains, as well as to asses for a selected strain, the response to the changes of physico-chemical variables (such as pH, NaCl and lipid content), in order to obtain predictive models describing their effects on the lipolysis pattern. The strains tested, having different environmental origin, showed different patterns of the free fatty acids (FFA) released. The clustering of the free fatty acids profiles evidenced that the unweighted average distance within the strains of the same species did not exceeded 30%. However, the lipolytic activity of some strains generated FFA profiles that differentiated from the majority of the strains considered. Also, when a single strain was inoculated in model systems in which pH, NaCl and milk fat were modulated according to a Central Composite Design (CCD), chemico-physical characteristics of the system led to marked variations in the lipolytic activity with consequent changes in individual fatty acids released. In most cases, when the same Y. lipolytica strain was used, under the experimental conditions adopted, the modulation of the lactic acid, NaCl and lipid content did not generate differences in the fatty acid release exceeding 20-21%. However, some combinations of factors remarkably affected lipase expression or activity, and generated differences in the fatty acid released higher than those observed among different strains of the same species.

Effect of trans-2-hexenal on the growth of Aspergillus flavus in relation to its concentration, temperature and water activity.

AIMS: The antifungal activity of trans-2-hexenal on Aspergillus flavus in a model system in relation to its concentration, incubation temperature and aw was assessed. METHODS AND RESULTS: A model describing the antifungal activity of the aldehyde in relation to these variables was obtained. CONCLUSION: According to this model, the inhibition of A. flavus was weakly dependent on the incubation temperature (at least within the range of values considered) and strongly affected by the trans-2-hexenal concentration and aw, which showed a remarkable synergistic effect. SIGNIFICANCE AND IMPACT OF THE STUDY: Trans-2-hexenal proved to be a molecule with remarkable antimicrobial properties, even when added in closed systems at low concentration.

Automated ribotyping using different enzymes to improve discrimination of Listeria monocytogenes isolates, with a particular focus on serotype 4b strains.

To develop improved automated subtyping approaches for Listeria monocytogenes, we characterized the discriminatory power of different restriction enzymes for ribotyping. When 15 different restriction enzymes were used for automated ribotyping of 16 selected L. monocytogenes isolates, the restriction enzymes EcoRI, PvuII, and XhoI showed high discriminatory ability (Simpson's index of discrimination > 0.900) and produced complete and reproducible restriction cut patterns. These three enzymes were thus evaluated for their ability to differentiate among isolates representing the two major serotype 4b epidemic clones, those having ribotype reference pattern DUP-1038 (51 isolates) and those having pattern DUP-1042 (20 isolates). Among these isolates, PvuII provided the highest discrimination for a single enzyme (nine different subtypes; index of discrimination = 0.518). A combination of PvuII and XhoI showed the highest discriminatory ability (index of discrimination = 0.590) for these isolates. A group of 44 DUP-1038 isolates and a group of 12 DUP-1042 isolates were identical to each other even when the combined data for all three enzymes were used. We conclude that automated ribotyping using different enzymes allows improved discrimination of L. monocytogenes isolates, including epidemic serotype 4b strains. We furthermore confirm that most of the isolates representing the genotypes linked to the two major epidemic L. monocytogenes clonal groups form two genetically homogeneous groups.

Effects of pH, temperature and NaCl concentration on the growth kinetics, proteolytic activity and biogenic amine production of Enterococcus faecalis.

In this work, the combined effects of temperature, pH and NaCl concentration on the growth dynamics of Enterococcus faecalis EF37, its proteolytic activity and its production of biogenic amines have been studied. The effects of the selected variables have been analysed using a Central Composite Design. The production of biogenic amines, under the adopted conditions, was found to be mainly dependent on the extent of growth of E. faecalis. Its proteolytic activity was not a limiting factor for the final amine production, because in the system studied (skim milk) an excess of precursors was guaranteed. Quantitatively, the most important biogenic amine produced was 2-phenylethylamine but substantial amounts of tyramine were detected in all the samples. This work confirms that the main biological feature influencing the biogenic amine formation is the extent of growth of microorganisms, like E. faecalis, characterised by decarboxylase activity. In the traditional and artisanal cheeses produced using raw milk, enterococci usually reach levels of 10(7) cells/g. With this perspective, it is important that the presence of biogenic amines due to the activities of these microorganisms is maintained within safe levels, without affecting the positive effects of enterococci on the final organoleptic characteristics of the cheese.

A survey of the enterococci isolated from an artisanal Italian goat's cheese (semicotto caprino).

Enterococci were isolated from semicotto caprino cheese, a traditional cheese produced in Southern Italy: they were a significant part of the microbial population of this cheese, confirming the importance of the presence of these micro-organisms during cheese-making and ripening. They were also identified and studied for their phenotypic and genotypic characteristics: Enterococcus faecalis and Ent. faecium were the most frequently isolated species, followed by Ent. durans, Ent. hirae and Ent. gallinarum. None of the isolates showed lipolytic activity, whereas they were characterized by a relevant proteolytic activity as well as an antagonistic activity towards Listeria innocua. One strain of Ent. gallinarum showed a low-level resistance to vancomycin, while six out of the 79 Ent. faecalis strains possessed beta-haemolysis reaction. The highest acidifying potential in skim milk was obtained by Ent. faecalis isolates. Thirty enterococcal strains representative of the different species at different ripening times were analysed by means of RAPD-PCR, and revealed species-specific profiles for all the considered species.

Effects of hexanal, trans-2-hexenal, and storage temperature on shelf life of fresh sliced apples.

In this paper, the effects of hexanal and trans-2-hexenal, which are both natural molecules characterizing apple aroma, on the microbial population and on color retention of fresh sliced apples were evaluated. In particular, a central composite design (CCD) was developed to assess the individual and interactive effects of the chosen volatile molecules and storage temperatures on (i) the growth of the naturally occurring microflora, (ii) the evolution over time of an inoculated spoilage yeast (Pichia subpelliculosa), and (iii) the enzymatic browning reaction in minimally processed apples. The inclusion of hexanal and trans-2-hexenal in the storage atmosphere of apple slices determined a significant extension of shelf life also when P. subpelliculosa was inoculated at levels of 10(3) colony-forming units/g and abusive storage temperatures were used. In fact, the presence of these molecules in the packaging atmospheres considerably prolonged the lag phases of the inoculated yeast and reduced the growth potential of naturally occurring bacteria. Moreover, the addition to the modified atmosphere of low levels of the hexanal increased the color stability of the products up to 16 days.