Dolphins and sea turtles may host zoonotic parasites and pathogenic bacteria as indicators of anthropic pressure in the Gulf of Taranto (Northern Ionian Sea, Central-Eastern Mediterranean Sea).

The occurrence of protozoan parasites Giardia duodenalis and Cryptosporidium spp. such as the pathogenic bacteria Salmonella spp. and Escherichia coli was molecularly investigated in the following free ranging species of striped dolphins (Stenella coeruleoalba), Risso's dolphins (Grampus griseus) as well as loggerhead (Caretta caretta) and green (Chelonia mydas) sea turtles living in the Gulf of Taranto (Mediterranean Sea). Out of forty-one investigated individuals belonging to the 4 species, 13 (31.7%) were positive to one or more pathogens and zoonotic G. duodenalis assemblage A, Cryptosporidium parvum and S. enterica were identified in striped dolphins, loggerhead and green sea turtles. In this work, the presence of these opportunistic pathogens has been investigated in fecal samples of free ranging dolphin and sea turtle species for the first time. Moreover, this is the first record of C. parvum in loggerhead sea turtles. These results may provide baseline data for the potential role of cetaceans and sea turtles as potential sentinel species for zoonotic and terrestrial pathogens in the marine environment.

Composting of Olive Mill Pomace, Agro-Industrial Sewage Sludge and Other Residues: Process Monitoring and Agronomic Use of the Resulting Composts.

The viability of co-composting of olive mill pomace added to sewage sludge with other organic residues was evaluated and the agronomic use of the final composts was investigated. Two composting piles at different carbon-nitrogen ratios were performed, in which olive mill pomace (OMP), sewage sludge from vegetable processing (SS), fresh residues from artichoke processing residues (AR), and wheat straw (WS) were used. The two composting piles were placed inside a specially built greenhouse and a turning machine pulled by a tractor was used for turning and shredding the organic matrix (every 6 days) during the process. The humidity and temperature of organic matrices have been monitored and controlled during the entire composting process, which lasted 90 days. The process was also monitored to evaluate the microbiological safety of the final compost. The humidity of both piles was always kept just above 50% until the end of the thermophilic phase and the maximum temperature was about 50 °C during the thermophilic phase. The carbon-nitrogen ratio decreased from 21.4 and 28.2, respectively (initial value at day 1 in Pile A and B), to values ranging from 12.9 to 15.1, both composts that originated from the two different piles were microbiologically safe. During a two-year period, the effects of different types of compost on the main qualitative parameters of processing tomato and durum wheat was evaluated. Five fertilization treatments were evaluated for tomato and durum wheat crops: unfertilized control (TR1); compost A (TR2); compost B (TR3); ½ mineral and ½ compost A (TR4); and mineral fertilizer commonly used for the two crops (TR5). Concerning the processing tomato yield, TR5 and TR4 showed the best results (2.73 and 2.51 kg, respectively). The same trend was observed considering the marketable yield per plant. The only difference was related to the treatments that included the compost (2.32, 1.77, and 1.73 kg/plant for TR4, TR3, and TR2, respectively). As regards the qualitative parameters of tomato, the highest average weight of the fruits was found in the TR5, TR4, and TR3 treatments (respectively, 73.67 g, 70.34 g, and 68.10 g). For durum wheat, only the protein component was differentiated between treatments. Furthermore, wheat grain yield parameters generally increased by combined application of mineral fertilizer and compost.

The Metabolism of Glucosinolates by Gut Microbiota.

Glucosinolates (GLS) and their derivatives are secondary plant metabolites abundant in Brassicaceae. Due to the enzymatic reaction between GLS and myrosinase enzyme, characteristic compounds with a pungent taste are formed, used by plants to defend themselves against insect herbivores. These GLS derivatives have an important impact on human health, including anti-inflammation and anti-cancer effects. However, GLS derivatives' formation needs previous enzymatic reactions catalyzed by myrosinase enzyme. Many of the brassica-based foods are processed at a high temperature that inactivates enzymes, hindering its bioavailability. In the last decade, several studies showed that the human gut microbiome can provide myrosinase activity that potentially can raise the beneficial effects of consumption of vegetables rich in GLS. The variability of the human gut microbiome (HGM) in human populations and the diverse intake of GLS through the diet may lead to greater variability of the real dose of pro-healthy compounds absorbed by the human body. The exploitation of the genetic and biochemical potential of HGM and correct ecological studies of both isolated strains and mixed population are of great interest. This review focuses on the most recent advances in this field.

Wild whale faecal samples as a proxy of anthropogenic impact.

The occurrence of protozoan parasite, bacterial communities, organic pollutants and heavy metals was investigated in free-ranging species of fin (Balaenoptera physalus, n. 2) and sperm (Physeter macrocephalus, n. 2) whales from the Pelagos Sanctuary, Corsican-Ligurian Provencal Basin (Northern-Western Mediterranean Sea). Out of four faecal samples investigated, two from fin whales and one from sperm whale were found positive to Blastocystis sp. A higher number of sequences related to Synergistetes and Spirochaetae were found in sperm whales if compared with fin whales. Moreover, As, Co and Hg were found exclusively in sperm whale faecal samples, while Pb was found only in fin whale faecal samples. The concentration of both PAH and PCB was always below the limit of detection. This is the first report in which the presence of these opportunistic pathogens, bacteria and chemical pollutants have been investigated in faecal samples of free-ranging whale species and the first record of Blastocystis in fin and sperm whales. Thus, this study may provide baseline data on new anthropozoonotic parasite, bacterial records and heavy metals in free-ranging fin and sperm whales, probably as a result of an increasing anthropogenic activity. This survey calls for more integrated research to perform regular monitoring programs supported by national and/or international authorities responsible for preservation of these still vulnerable and threatened whale species in the Mediterranean Sea.

The glucosinolates and their bioactive derivatives in Brassica: a review on classification, biosynthesis and content in plant tissues, fate during and after processing, effect on the human organism and interaction with the gut microbiota.

The present study is a systematic review of the scientific literature reporting content, composition and biosynthesis of glucosinolates (GLS), and their derivative compounds in Brassica family. An amended classification of brassica species, varieties and their GLS content, organized for the different plant organs and in uniformed concentration measure unit, is here reported for the first time in a harmonized and comparative manner. In the last years, the studies carried out on the effect of processing on vegetables and the potential benefits for human health has increased rapidly and consistently the knowledge on the topic. Therefore, there was the need for an updated revision of the scientific literature of pre- and post-harvest modifications of GLS content, along with the role of gut microbiota in influencing their bioavailability once they are ingested. After analyzing and standardizing over 100 articles and the related data, the highest GLS content in Brassica, was declared in B. nigra (L.) W. D. J. Koch (201.95 ± 53.36 µmol g-1), followed by B. oleracea Alboglabra group (180.9 ± 70.3 µmol g-1). The authors also conclude that food processing can influence significantly the final content of GLS, considering the most popular methods: boiling, blanching, steaming, the latter can be considered as the most favorable to preserve highest level of GLS and their deriviatives. Therefore, a mild-processing strategic approach for GLS or their derivatives in food is recommended, in order to minimize the loss of actual bioactive impact. Finally, the human gut microbiota is influenced by Brassica-rich diet and can contribute in certain conditions to the increasing of GLS bioavailability but further studies are needed to assess the actual role of microbiomes in the bioavailability of healthy glucosinolate derivatives.

Antibiotic-Resistant Bacteria in Greywater and Greywater-Irrigated Soils.

This study represents the first systematic attempt to evaluate antibiotic-resistant bacteria (ARB) occurrence in treated greywater and the potential spread of these bacteria from the greywater to greywater-irrigated soil. Treated greywater from three recirculating vertical flow constructed wetlands, each located in a household in the central Negev Desert, Israel, was surveyed. The presence of antibiotic-resistant bacteria in raw and treated greywater was investigated with culture and molecular methods, as well as their presence in the corresponding treated-greywater-irrigated soils. Additionally, the effectiveness of chlorination to prevent the spread of ARB was tested. The total count of tetracycline-resistant bacteria significantly increased in the treated greywater, likely due to their concentration on the filter matrix of the treatment systems. Twenty-four strains of tetracycline-resistant bacteria were isolated and identified at the genus level by 16Sr RNA gene sequencing. All the tetracycline-resistant bacteria showed high resistance traits, and some of them presented multiple antibiotic resistances. Six tetracycline resistance genes (coding for efflux and ribosomal resistance mechanisms) and five ß-lactamase genes were detected. In 14 of the isolated strains, the gene tet39, which is phylogenetically related to both environmental and clinical strains, was identified. All the tet39 resistant bacteria were positive to at least one of the ß-lactamase genes tested. Chlorination was found to be an efficient method to reduce ARB in treated greywater. We concluded that disinfection of treated greywater may reduce the risks not only from the potential presence of pathogens but also from the presence of ARB and antibiotic resistance genes.

Metataxonomy and functionality of wood-tar degrading microbial consortia.

Wood-tar is a liquid material obtained by wood gasification process, and comprises several polycyclic aromatic hydrocarbons (PAH). Tar biodegradation is a very challenging task, due to its toxicity and to its complex chemistry. The 'microbial resource management' concerns the use of environmental microbial communities potentially able to provide us services. We applied this concept in tar biodegradation. Tar composed by several PAH (including phenanthrene, acenaphthylene and fluorene) was subjected to a biodegradation process in triplicate microcosms spiked with a microbial community collected from PAH-rich soils. In 20 days, 98.9% of tar was mineralized or adsorbed to floccules, while negative controls showed poor PAH reduction. The dynamics of fungal and bacterial communities was assessed through Automated Ribosomal Intergenic Spacer Analysis (ARISA), 454 pyrosequencing of the fungal ITS and of the bacterial 16S rRNA. Quantification of the degrading bacterial communities was performed via quantitative Real Time PCR of the 16S rRNA genes and of the cathecol 2,3-dioxygenase genes. Results showed the importance of fungal tar-degrading populations in the first period of incubation, followed by a complex bacterial dynamical growth ruled by co-feeding behaviors.

Impact of the reusing of food manufacturing wastewater for irrigation in a closed system on the microbiological quality of the food crops.

In order to evaluate if the reuse of food industry treated wastewater is compatible for irrigation of food crops, without increased health risk, in the present study a cropping system, in which ground water and treated wastewater were used for irrigation of tomato and broccoli, during consecutive crop seasons was monitored. Water, crop environment and final products were monitored for microbial indicators and pathogenic bacteria, by conventional and molecular methods. The microbial quality of the irrigation waters influenced sporadically the presence of microbial indicators in soil. No water sample was found positive for pathogenic bacteria, independently from the source. Salmonella spp. and Listeria monocytogenes were detected in soil samples, independently from the irrigation water source. No pathogen was found to contaminate tomato plants, while Listeria monocytogenes and E. coli O157:H7 were detected on broccoli plant, but when final produce were harvested, no pathogen was detected on edible part. The level of microbial indicators and detection of pathogenic bacteria in field and plant was not dependent upon wastewater used. Our results, suggest that reuse of food industry wastewater for irrigation of agricultural crop can be applied without significant increase of potential health risk related to microbial quality.

Autochthonous lactic acid bacteria with probiotic aptitudes as starter cultures for fish-based products.

This study focused on the selection of lactic starters with probiotic properties for the production of fermented fish-products by the use of a multivariate approach (Cluster Analysis and Principal Component Analysis). Seventy-five isolates were recovered from fish intestinal microbiota and characterized by evaluating phenotypical, technological and probiotic traits; the most promising isolates were molecularly identified and then used into fish fermented sausage production. Namely, data from technological characterization were modelled through Growth Index and used as input to run a preliminary selection. Thus, 15 promising strains were selected and subjected to probiotic characterization; considering the results from probiotic tests, 3 promising strains were finally chosen (11, 68 and 69), identified as members of the genus Lactobacillus and used for the validation at laboratory level through the assessment of their performances for the production of fermented fish sausages. The results were promising as the use of the selected strains reduced the fermentation time (2 days) ensuring a good microbiological quality of the final product.

Inoculum pre-treatment affects the fermentative activity of hydrogen-producing communities in the presence of 5-hydroxymethylfurfural.

To enhance the productivity of mixed microbial cultures for fermentative bio-hydrogen production, chemical-physical pre-treatments of the original seed are needed to suppress the activity of hydrogen (H2)-consuming microbes. This approach might influence negatively the composition and diversity of the hydrogen-producing community with consequences on the functional stability of the H2-producing systems in case of perturbations. In this study, we aimed at investigating the effect of different types of pre-treatment on the performance of hydrogen production systems in the presence of an inhibitor, such as 5-hydroxymethylfurfural (HMF). The efficiency and the microbial community structure of batch reactors amended with HMF and inoculated with non-pretreated and pretreated (acid, heat shock, and aeration) anaerobic sludge were evaluated and compared with control systems. The type of pre-treatments influenced the microbial community assembly and activity in inhibited systems, with significant effect on the performance. Cumulative H2 production tests showed that the pre-aerated systems (control and HMF inhibited) were the most efficient, while the difference of the lag phase of the pre-acidified control and HMF-added test was negligible. Analyses of the structure of the enriched microbial community in the systems through PCR-denaturing gradient gel electrophoresis (DGGE) followed by band sequencing revealed that the differences in performance were mostly related to shifts in the metabolic pathways rather than in the predominant species. In conclusion, the findings suggest that the use of specific inoculum pre-treatment could contribute to regulate the metabolic activity of the fermentative H2-producing bacteria in order to enhance the bio-energy production.

A preliminary and qualitative study of resource ratio theory to nitrifying lab-scale bioreactors.

The incorporation of microbial diversity in design would ideally require predictive theory that would relate operational parameters to the numbers and distribution of taxa. Resource ratio-theory (RRT) might be one such theory. Based on Monod kinetics, it explains diversity in function of resource-ratio and richness. However, to be usable in biological engineered system, the growth parameters of all the bacteria under consideration and the resource supply and diffusion parameters for all the relevant nutrients should be determined. This is challenging, but plausible, at least for low diversity groups with simple resource requirements like the ammonia oxidizing bacteria (AOB). One of the major successes of RRT was its ability to explain the 'paradox of enrichment' which states that diversity first increases and then decreases with resource richness. Here, we demonstrate that this pattern can be seen in lab-scale-activated sludge reactors and parallel simulations that incorporate the principles of RRT in a floc-based system. High and low ammonia and oxygen were supplied to continuous flow bioreactors with resource conditions correlating with the composition and diversity of resident AOB communities based on AOB 16S rDNA clone libraries. Neither the experimental work nor the simulations are definitive proof for the application of RRT in this context. However, it is sufficient evidence that such approach might work and justify a more rigorous investigation.

Selection of autochthonous strains as starter cultures for fermented fish products.

This paper was the 1st research focusing on the design of a halophilic lactic starter for the production of fermented fish products using a quantitative approach, based on the evaluation of the growth index and acidification score, as well as on the use of a multivariate approach to select the most promising strains. Fifty-nine strains were randomly selected from salted fish and phenotypically characterized through Gram staining, catalase activity, glucose metabolism, H2 S and indole production, nitrate reduction, citrate utilization, and hydrolysis of arginine, esculin, casein, gelatin, starch, Tween 80, and urea. Then the Gram positive isolates (44 out of 59) were studied for their growth at different temperatures (10, 25, 40, and 55 °C), salt (0%, 20%, and 30%), pHs (4.5 and 9.5), and acidification score in lab medium. Data were modelled through growth index and used as input to run a preliminary cluster analysis and a principal component analysis. Three promising strains were selected, identified as members of the genus Pediococcus and used for the validation at laboratory level through the assessment of their performances in the production of a fermented fish sauce. The results were really promising as their use not only reduced the fermentation time (2 d) but also improved the microbiological quality of the final product. This paper represents a 1st report on the use of a simple step-by-step methodology to select promising halophilic strains for the optimization of a starter for fish-fermented products.

A fast, reliable, and sensitive method for detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in ready-to-eat fresh-cut products by MPN-qPCR.

In the present work we developed a MPN quantitative real-time PCR (MPN-qPCR) method for a fast and reliable detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in minimally processed vegetables. In order to validate the proposed technique, the results were compared with conventional MPN followed by phenotypic and biochemical assays methods. When L. monocytogenes and E. coli O157:H7 were artificially inoculated in fresh-cut vegetables, a concentration as low as 1 CFU g(-1) could be detected in 48 hours for both pathogens. qPCR alone allowed a limit of detection of 10(1) CFU g(-1) after 2 hours of enrichment for L. monocytogenes and E. coli O157:H7. Since minimally processed ready-to-eat vegetables are characterized by very short shelf life, our method can potentially address the consistent reduction of time for microbial analysis, allowing a better management of quality control. Moreover, the occurrences of both pathogenic bacteria in mixed salad samples and fresh-cut melons were monitored in two production plants from the receipt of the raw materials to the early stages of shelf life. No sample was found to be contaminated by L. monocytogenes. One sample of raw mixed salad was found positive to an H7 enterohemorrhagic serotype.

Selection of yeasts as starter cultures for table olives.

Ninety-nine yeasts were isolated from Bella di Cerignola table olives; first, the strains were studied in relation to their ability to produce biogenic amines in a laboratory medium and 49 strains were positive to this assay and cut off from the research. The remaining 50 strains were characterized for their enzymatic traits (ß-glucosidase, catalase, pectolytic, xylanolytic, and lipolytic activities) and for their ability to grow at different temperatures, pHs, with salt or lactic/acetic acids added. Data were used for the evaluation of growth index and submitted to cluster and principal component analyses to choose the most promising 4 strains. In the final step of the research, the strains were inoculated as a cocktail in a model brine, containing different amounts of salt (4% to 12%) and glucose (0% to 3%), and adjusted to different pHs (4.0 to 9.0). Data analysis through a multiple regression procedure highlighted that salt, glucose, and pH acted in a different way within the storage and NaCl affected yeast growth only for few days, and then glucose and pH played a major role.

Ochratoxin A removal by Saccharomyces cerevisiae strains: effect of wine-related physicochemical factors.

BACKGROUND: This study investigated the effect of some physicochemical parameters on the removal of ochratoxin A (OTA) by yeasts. RESULTS: Two wild strains of Saccharomyces cerevisiae (W47 and Y28) were used to assess OTA removal under various conditions of temperature, pH, ethanol content and incubation time. All samples were analysed for OTA concentration by enzyme-linked immunosorbent assay (ELISA). In addition, yeast oenological traits were investigated: qualitative and technological traits were assessed on appropriate laboratory media, while the main products of microfermentation (sugars, ethanol, glycerol, acetic acid) were evaluated by Fourier transform infrared spectroscopy (FTIR). The results showed OTA reduction by 36-42% in cultures containing 100 g L⁻¹ ethanol incubated at pH 3.5 and 37 °C. CONCLUSION: OTA removal was affected by contact time, pH and ethanol content, as it was increased at low pH and by 100 g L⁻¹ ethanol. Moreover, the phenomenon was reversible, as OTA was lowest after 4 days, then it was partially released in the medium.

Determination of biogenic amines in wine by thin-layer chromatography/densitometry.

Biogenic amines are small-molecular-weight organic bases that can be encountered in all fermented foods, including wine. Ingestion of wine containing biogenic amines, and especially histamine, can result in health nuisances. HPLC is the analytical technique most often employed in the determination of biogenic amines in wine but HPLC-based methods are expensive and time-consuming. A new method, based upon amine dansylation and TLC/densitometry, was developed and validated. This allowed for the determination of histamine, tyramine, putrescine and cadaverine in wine at concentrations between 1 and 20mg/L. Analytical performances adequately complied with the needs of routine wine analysis, moreover the method was high-throughput and inexpensive. A simpler, semi-quantitative version of the method, based on visual evaluation of spot intensity, was also developed.

Isolation and characterization of tyramine-producing Enterococcus faecium strains from red wine.

Enterococcus faecium strains were isolated from red wines undergoing malolactic fermentation and identified by comparison of their 16S rDNA gene sequences with those included in the GenEMBL Databases. The tyrosine decarboxylase gene was identified in all the strains analysed by PCR using gene-specific primers and the ability to produce tyramine in a synthetic media was analysed by RP-HPLC. Survival of an E. faecium strain was also evaluated in microvinification assays using two different musts with different ethanol concentrations (10% and 12% (v/v)). Tyramine production was monitored during the vinification trials. Our results suggest that E. faecium strains isolated from wine are able to produce tyramine and tolerate wine conditions following a pre-acidic stress.

Occurrence and characterization of Escherichia coli O157 and other serotypes in raw meat products in Morocco.

In this study, 100 raw meat samples were collected from 15 local Moroccan butcheries in five different areas of the city of Rabat during a period of 4 months. Overall, 7 of 15 butcheries from three areas of the city yielded strains of Escherichia coli O157. Single isolates from 9 (9%) of 100 raw meat samples were biochemically and serologically confirmed as E. coli O157. Using molecular techniques, two strains were positive for the Shiga toxin, with two additional strains containing an attaching-effacing gene. All potentially virulent serotypes isolated from these meat samples showed distinct pulsed-field gel electrophoresis profiles. Based on antibiotic susceptibility testing, more than 70% of the isolates were resistant to ampicillin and clavulanic acid-amoxicillin. Moreover, one strain was resistant to more than three antibiotics. Our study represents the first survey of E. coli O157 and related serotypes in raw meat products in Morocco.