High-quality genome sequence assembly of R.A73 Enterococcus faecium isolated from freshwater fish mucus.

BACKGROUND: Whole-genome sequencing using high throughput technologies has revolutionized and speeded up the scientific investigation of bacterial genetics, biochemistry, and molecular biology. Lactic acid bacteria (LABs) have been extensively used in fermentation and more recently as probiotics in food products that promote health. Genome sequencing and functional genomics investigations of LABs varieties provide rapid and important information about their diversity and their evolution, revealing a significant molecular basis. This study investigated the whole genome sequences of the Enterococcus faecium strain (HG937697), isolated from the mucus of freshwater fish in Tunisian dams. Genomic DNA was extracted using the Quick-GDNA kit and sequenced using the Illumina HiSeq2500 system. Sequences quality assessment was performed using FastQC software. The complete genome annotation was carried out with the Rapid Annotation using Subsystem Technology (RAST) web server then NCBI PGAAP. RESULTS: The Enterococcus faecium R.A73 assembled in 28 contigs consisting of 2,935,283 bps. The genome annotation revealed 2884 genes in total including 2834 coding sequences and 50 RNAs containing 3 rRNAs (one rRNA 16 s, one rRNA 23 s and one rRNA 5 s) and 47 tRNAs. Twenty-two genes implicated in bacteriocin production are identified within the Enterococcus faecium R.A73 strain. CONCLUSION: Data obtained provide insights to further investigate the effective strategy for testing this Enterococcus faecium R.A73 strain in the industrial manufacturing process. Studying their metabolism with bioinformatics tools represents the future challenge and contribution to improving the utilization of the multi-purpose bacteria in food.

In vitro probiotic profiling of novel Enterococcus faecium and Leuconostoc mesenteroides from Tunisian freshwater fishes.

Novel lactic acid bacteria isolated from different organs of freshwater fish were examined for their potential application as probiotics in raw and processed foods. Four isolates of Enterococcus faecium and Leuconostoc mesenteroides were identified at the molecular level by 16S rRNA sequencing and random amplification of polymorphic DNA - polymerase chain reaction, and their antimicrobial activity against a panel of pathogens and food-poisoning bacteria was investigated. The whole bacteriocins of the 4 isolates were characterized by enterobacterial repetitive intergenic consensus sequences in PCR. The isolates exhibited high inhibitory activities against food-borne pathogens and spoilage microbial species and have significant probiotic profiles, since they survived at pH 3.0 and in the presence of bile salts, pancreatin, and pepsin, without any detectable hemolytic activity. Further, moderate heat resistance, adhesion ability to steel surfaces, and sensitivity to clinically relevant antimicrobial agents were revealed for all the isolates. These results highlight the specific probiotic properties of the strains and give evidence for potential application in minimally processed foods subjected to moderate heat processing.

Foraminiferal biotopes and their distribution control in Ria de Aveiro (Portugal): a multiproxy approach.

Ria de Aveiro, which is located in the centre of Portugal (40° 38' N, 8° 45' W), is a well-mixed and complex coastal lagoon that is separated from the sea by a sandy barrier and connects with the Atlantic through an artificial inlet. Tidal currents are the main factor controlling the lagoon's hydrodynamics and, to a great extent, the sedimentary dynamic. The inner lagoonal zones receive input from several rivers and experience the pressure caused by the accumulation of organic matter and pollutants (namely, trace metals) from diverse anthropic activities. This paper is the first piece of work aiming to recognize, characterize and explain the main benthic foraminiferal biotopes in Ria de Aveiro. To provide a broad overview of this kind of setting, our results are compared to those of previous published studies conducted in similar transitional environments. The research is based on an investigation of 225 sites spread throughout this ecosystem. Utilizing a statistical approach, this study analyses the details of dead benthic foraminiferal assemblages composed of 260 taxa, the texture and composition (mineralogical and geochemical) of the sediment and physicochemical data. On the basis of the results of R-mode and Q-mode cluster analyses, several different biotopes can be defined as marsh biotope/near-marsh biotope; marginal urban/marginal urban mixing biotope; inner-outer lagoon biotope or enclosed lagoon; outer lagoon biotope, mixed sub-biotope; and outer lagoon, marine sub-biotope. These biotopes are related to foraminifera assemblages and substrate type and are influenced by local currents, water depth, chemical and physicochemical conditions, river or oceanic proximity, and anthropogenic impact, as evidenced by the mapping of the six factor loadings of the principal component analysis conducted herein. Based on a similar methodology of analysis as that applied in previous studies in the Lagoon of Venice, comparable biotypes were identified in Lagoon of Aveiro.

Detection and quantification of microplastics in Posidonia oceanica banquettes in the Gulf of Gabes, Tunisia.

Plastic pollution and microplastic (MP) debris are some of the most significant solid waste pollutants, threatening the marine environment and causing sediment accumulation. Coastal seagrass areas are usually important habitats that support multiple living species and provide several ecosystem services. This study aimed to determine the abundance, characteristics, and composition of microplastics on the southern side of the Tunisian Mediterranean Sea by using Posidonia oceanica (P. oceanica) as a crucial trap for microplastics. Samples of Posidonia leaves were collected from the Tunisian coastal area of Gabes-City. The characterization of microplastic detritus was carried out by stereomicroscopy, and acid digestion of Posidonia tissue leaves was performed for qualitative and quantitative analysis of MPs using NMR spectroscopy. The study revealed pellets, threads, and fragments of polymers as the frequent forms found in MPs. Polyethylene, polystyrene, and bis(2-ethyl-hexyl) phthalates were the most abundant materials detected. P. oceanica leaves contributed notably to microplastic subsidence, seafloor horizontal migration, and sediment burial. Thus, marine flora appeared to be a good tool to detect and monitor plasticizers, and further studies of the P. oceanica seagrass areas will help in developing a more comprehensive knowledge of chemicals spreading over a geographical zone. The results obtained will be used for developing baseline data on plasticizer contamination on the wide-ranging marine coast.

Phylogenetic analysis of antimicrobial lactic acid bacteria from farmed seabass Dicentrarchus labrax.

The use of lactic acid bacteria (LAB) in the prevention or reduction of fish diseases is receiving increasing attention. In the present study, 47 LAB strains were isolated from farmed seabass ( Dicentrarchus labrax ) and were phenotypically and phylogenetically analysed by 16S rDNA and randomly amplified polymorphic DNA - polymerase chain reaction (RAPD-PCR). Their antimicrobial effect was tested in vitro against a wide variety of pathogenic and spoilage bacteria. Most of the strains isolated were enterococci belonging to the following species: Enterococcus faecium (59%), Enterococcus faecalis (21%), Enterococcus sanguinicola (4 strains), Enterococcus mundtii (1 strain), Enterococcus pseudoavium (1 strain), and Lactococcus lactis (1 strain). An Aerococcus viridans strain was also isolated. The survey of their antimicrobial susceptibility showed that all isolates were sensitive to vancomycin and exhibited resistance to between 4 and 10 other antibiotics relevant for therapy in human and animal medicine. Different patterns of resistance were noted for skin and intestines isolates. More than 69% (32 strains) of the isolates inhibited the growth of the majority of pathogenic and spoilage bacteria tested, including Listeria monocytogenes, Staphylococcus aureus, Aeromonas hydrophila, Aeromonas salmonicida, Vibrio anguillarum, and Carnobacterium sp. To our knowledge, this is the first report of bioactive enterococcal species isolated from seabass that could potentially inhibit the undesirable bacteria found in food systems.

Bacterial community structure of sediments of the bizerte lagoon (Tunisia), a southern Mediterranean coastal anthropized lagoon.

In order to estimate how pollution affects the bacterial community structure and composition of sediments, chemical and molecular approaches were combined to investigate eight stations around the Bizerte lagoon. Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified 16S rRNA genes revealed that each station was characterized by a specific bacterial community structure. The combination of this data with those of chemical analysis showed a correlation between the bacterial fingerprint and the pollutant content, principally with hydrocarbon pollution. The composition of the bacterial community of two contrasted stations related to the pollution revealed sequences affiliated to alpha, beta, gamma, delta, epsilon subclass of the Proteobacteria, Actinobacteria, and Acidobacteria in both stations although in different extent. Gamma and delta subclass of the Proteobacteria were dominant and represent 70% of clones in the heavy-metal-contaminated station and 47% in the polyaromatic hydrocarbon (PAH)-contaminated. Nevertheless, most of the sequences found were unaffiliated to cultured bacteria. The adaptation of the bacterial community mainly to PAH compounds demonstrated here and the fact that these bacterial communities are mainly unknown suggest that the Bizerte lagoon is an interesting environment to understand the capacity of bacteria to cope with some pollutants.

Seasonal ecotoxicological monitoring of freshwater zooplankton in Bir Mcherga dam (Tunisia).

Dams represent large semi-closed reservoirs of pesticides and various organic and inorganic pollutants from agricultural and human activities, and their monitoring should receive special attention. This study evaluated the environmental health status of Bir Mcherga dam using zooplankton species. The dam has a capacity of 130 Mm3 and its waters are used for irrigation, water drinking supply, and fishery. Copepods and cladocerans (crustaceans) were collected in situ monthly between October and August 2012. Oxidative stress (CAT, MDA), neurotoxicity (AChE), and genotoxicity (micronucleus test) biomarkers were analyzed in two zooplankton species: Acanthocyclops robustus and Diaphanosoma mongolianum. High values of cells with a micronucleus were observed during summer. AChE activities were inhibited during early winter and summer. The high seasonal variability of CAT and MDA levels indicates that zooplankton is continuously exposed to different oxidative stresses. These results suggest that there is an obvious and continuous multi-faceted stress in Bir Mcherga reservoir and, consequently, an urgent monitoring of freshwater environments in Tunisia is needed, particularly those intended for human consumption and irrigation.

Crystal Structure, Hirshfeld Surface Analysis and Biological Activities of trans-Dipyridinebis (3-Acetyl-2-Oxo-2H-Chromen-4-Olato)Cobalt(II).

The novel cobalt(II) complex, trans-dipyridinebis(3-acetyl-2-oxo-2H-chromen-4-olato)cobalt(II), was synthesized in ethanol. The coordination sphere of the cobalt cation was elucidated using single-crystal X-ray diffraction analysis and spectroscopic techniques (FT-IR, UV-Visible and fluorescence). Hirshfeld surface analysis indicates that hydrogen bond interactions, such as C-H···O hydrogen bonding between the oxygen of lactone group and the pyridine appear as a primary interaction between the complex's molecules. The presence of π- π stacking was evident by the shape index and curvature. Analysis of 2D fingerprint plots confirm that intermolecular H···H, C···H and H···O interactions are well dominated and are in complement to the Hirshfeld surface. The metal-ligand coordination strongly influences the fluorescence intensity (the fluorescence quenching) and the offset of the emission wavelength. The metal complex was monitored for antimicrobial activity using the disk diffusion method and showed significant activity compared to the coumarin ligand.

Antimicrobial Fatty Acids from Green Alga Ulva rigida (Chlorophyta).

This study deals with the antimicrobial potential assessment of Ulva rigida, in regard to collection period and sampling site. Besides, we assess the chemical composition of bioactive compounds. For this purpose, Ulva rigida was seasonally collected from two northern sites in Tunisia, Cap Zebib rocky shore (CZ) and Ghar El Melh lagoon (GEM). Crude organic extracts were prepared using dichloromethane and dichloromethane/methanol and tested against 19 indicator microorganisms using the disk diffusion method and microdilution technique to determine the minimum inhibitory concentration (MIC). Silica gel column and thin layer chromatography were used for purification of active compounds. Nuclear magnetic resonance (NMR) and gas chromatography were used for compounds identification. Samples of Ulva rigida collected from the two sites have uniform antimicrobial activity throughout the year. Algae collected from the lagoon showed the largest spectrum of activity and were used for subsequent analysis. Bioguided purification of extracts from Ulva rigida, collected at GEM, leads to 16 active fractions with antibacterial effect mainly against Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212. These fractions were identified as fatty acids, mainly oleic (C18: 1 w9), linoleic (C18: 2 w6), palmitic (C16: 0), and stearic (C14: 0). MICs values ranged from 10 to 250 µg/ml.

Trace element accumulation and elutriate toxicity in surface sediment in northern Tunisia (Tunis Gulf, southern Mediterranean).

Metal concentrations in sediments were investigated in the Gulf of Tunis, Tunisia, in relation to anthropic activities along the Mejerda River and Ghar El Melh Lagoon, with effluents discharged into the gulf. Distribution of grain size showed that the silty fraction is dominant with 53%, while sand and clay averages are 34 and 12% respectively. Zn concentration increased in the vicinity of the Mejerda River while Pb was at its highest levels at the outlet of Ghar El Mehl Lagoon. Sediment elutriate toxicity, as measured by oyster embryo bioassays, ranged from 10 to 45% abnormalities after 24h, but no relation was found between metal concentration and sediment toxicity. The AVS fraction that represents monosulfide concentrations in the sediment was higher in the central part of the gulf than in the coastal zone. The results reveal the influence of AVS, TOC and grain size on metal speciation and sediment toxicity.

Antimicrobial Activities of Bacteria Associated with the Brown Alga Padina pavonica.

Macroalgae belonging to the genus Padina are known to produce antibacterial compounds that may inhibit growth of human- and animal pathogens. Hitherto, it was unclear whether this antibacterial activity is produced by the macroalga itself or by secondary metabolite producing epiphytic bacteria. Here we report antibacterial activities of epiphytic bacteria isolated from Padina pavonica (Peacocks tail) located on northern coast of Tunisia. Eighteen isolates were obtained in pure culture and tested for antimicrobial activities. Based on the 16S rRNA gene sequences the isolates were closely related to Proteobacteria (12 isolates; 2 Alpha- and 10 Gammaproteobacteria), Firmicutes (4 isolates) and Actinobacteria (2 isolates). The antimicrobial activity was assessed as inhibition of growth of 12 species of pathogenic bacteria (Aeromonas salmonicida, A. hydrophila, Enterobacter xiangfangensis, Enterococcus faecium, Escherichia coli, Micrococcus sp., Salmonella typhimurium, Staphylococcus aureus, Streptococcus sp., Vibrio alginoliticus, V. proteolyticus, V. vulnificus) and one pathogenic yeast (Candida albicans). Among the Firmicutes, isolate P8, which is closely related to Bacillus pumilus, displayed the largest spectrum of growth inhibition of the pathogenic bacteria tested. The results emphasize the potential use of P. pavonica associated antagonistic bacteria as producers of novel antibacterial compounds.

Assessing pollution in a Mediterranean lagoon using acid volatile sulfides and estimations of simultaneously extracted metals.

Bizerte Lagoon is a southern Mediterranean semi-enclosed lagoon with a maximum depth of 12 m. After assessing sediment quality, the authors report on the physicochemical characteristics of the lagoon's surface sediment using SEM (simultaneously extracted metals) and AVS (acid volatile sulfides) as proxies. Biogeochemical tools are used to investigate the environmental disturbance at the water-sediment interface by means of SEM and AVS to seek conclusions concerning the study area's pollution status. Results confirm accumulation of trace elements in sediment. The use of the SEM-AVS model with organic matter in sediment (ƒOC) confirms possible bioavailability of accumulated trace elements, especially Zn, in the southern part of the lagoon, with organic matter playing an important role in SEM excess correction to affirm a nontoxic total metal sediment state. Individual trace element toxicity is dependent on the bioavailable fraction of SEMMetal on sediment, as is the influence of lagoon inflow from southern water sources on element bioavailability. Appropriate management strategies are highly recommended to mitigate any potential harmful effects on health from this heavy-metal-based pollution.

Organic matter quantity and quality, metals availability and foraminiferal assemblages as environmental proxy applied to the Bizerte Lagoon (Tunisia).

This study analyzes the benthic trophic state of Bizerte Lagoon (Tunisia) based on the total organic matter and the bioavailability of biopolymeric carbon including proteins (PTN), carbohydrates (CHO), lipids (LIP), chlorophyll a, as well as bacteria counts. The overall simultaneously extracted metals (SEM), and acid volatile sulfides (AVS) as well as the SEM/AVS ratio indicative of the toxicity of the sediments also were analyzed aiming to study their impact in the dimension, composition and structure of both dead and living benthic foraminiferal assemblages. In the studied sites TOC content is relatively high and the PTN/CHO values indicate that they can be considered as meso-eutrophic environments. The CHO/TOC and C/N values suggest that the OM which accumulated on the sediments surface has mainly natural origin despite the introduction of municipal and industrial effluents in the lagoon and the large bacterial pool. The living assemblages of benthic foraminifera of Bizerte Lagoon are quite different to other Mediterranean transitional systems studied until now. They are composed of typical lagoonal species but also include several marine and opportunistic species including significant numbers of bolivinids, buliminids, Nonionella/Nonionoides spp. and Cassidulina/Globocassidulina spp. These assemblages seem to benefitfrom the physicochemical parameters and the sediment stability. They may however face environmental stress in the lagoon related to the AVS production as a result of the organic matter degradation and toxicity in some areas due to the available concentrations of metals. Nonetheless statistical results evidence that the structure and dimension of assemblages are being controlled mostly by OM quantity and quality related mainly to the availability of PTN, CHO and chlorophyll a. Results of this work support the importance of considering OM quantity and quality in studies of environmental impact in coastal systems.

Environmental Quality Assessment of Bizerte Lagoon (Tunisia) Using Living Foraminifera Assemblages and a Multiproxy Approach.

This study investigated the environmental quality of the Bizerte Lagoon (Tunisia) through an integrated approach that combined environmental, biogeochemical, and living benthic foraminiferal analyses. Specifically, we analyzed the physicochemical parameters of the water and sediment. The textural, mineralogical, and geochemical characteristics of the sediment, including total organic carbon, total nitrogen, simultaneously extracted metals (SEM), acid volatile sulfides (AVS), chlorophyll a, CaCO3, and changes in bacterial populations and carbon isotopes were measured. The SEM/AVS values indicated the presence of relatively high concentrations of toxic metals in only some areas. Foraminiferal assemblages were dominated by species such as A. parkinsoniana (20-91%), Bolivina striatula (<40%), Hopkinsina atlantica (<17%), and Bolivina ordinaria (<15%) that cannot be considered typical of impacted coastal lagoons both in Mediterranean and northeast Atlantic regions. The results of this work suggest that Bizerte Lagoon is a unique setting. This lagoon is populated by typical marine species that invaded this ecosystem, attracted not only by the prevailing favorable environmental conditions but also by the abundance and quality of food. The results indicate that the metal pollution found in some areas have a negative impact on the assemblages of foraminifera. At present, however, this negative impact is not highly alarming.

Accumulation of trace metals in sediments in a Mediterranean Lagoon: Usefulness of metal sediment fractionation and elutriate toxicity assessment.

The authors investigated sediment quality in Bizerte Lagoon (Tunisia) focusing on geochemical characteristics, metal sediment fractionation and elutriate toxicity assessment. Nickel, Cu, Zn, Pb, Cr and Cd partitioning in sediments was studied; accumulation and bioavailability were elucidated using enrichment factors, sequential extractions, redox potential, acid volatile sulfide and biotest procedures in toxicity evaluation. Results revealed an accumulation for Pb and Zn, reaching 99 and 460 mg kg(-1) respectively. In addition, the acid volatile sulfide values were high in both eastern and western lagoon areas, thus affecting metal availability. Mean enrichment factor values for Pb and Zn were 4.8 and 4.9, respectively, with these elements as the main contributors to the lagoon's moderate enrichment level. Toxicity levels were influenced by accumulation of Zn in different surface sediment areas. Core sediments were investigated in areas with the highest metal concentrations; metal fractionation and biotest confirmed that Zn contributes to sediment toxicity.

Dynamic energy budget model: a monitoring tool for growth and reproduction performance of Mytilus galloprovincialis in Bizerte Lagoon (Southwestern Mediterranean Sea).

Mussel farming is the main economic activity in Bizerte Lagoon, with a production that fluctuates depending on environmental factors. In the present study, we apply a bioenergetic growth model to the mussel Mytilus galloprovincialis, based on dynamic energy budget (DEB) theory which describes energy flux variation through the different compartments of the mussel body. Thus, the present model simulates both mussel growth and sexual cycle steps according to food availability and water temperature and also the effect of climate change on mussel behavior and reproduction. The results point to good concordance between simulations and growth parameters (metric length and weight) for mussels in the lagoon. A heat wave scenario was also simulated using the DEB model, which highlighted mussel mortality periods during a period of high temperature.

Discovery of novel biopreservation agents with inhibitory effects on growth of food-borne pathogens and their application to seafood products.

Selection of protective cultures is relevant in order to biopreserve and improve the functional safety of food products, mainly through inhibition of spoilage and/or pathogenic bacteria. Accordingly, the present study investigated potential applications of lactic acid bacteria (LAB) in the biopreservation of fish and shellfish products. For this purpose, a collection of 84 LAB strains isolated from sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) was identified and characterized for their inhibitory activities against the most relevant seafood-spoilage and pathogenic bacteria potentially present in commercial products. The bioactive strains belonged to the genus Enterococcus and exhibited inhibition against Carnobacterium sp, Bacillus sp, Listeria monocytogenes, Aeromonas salmonicida, Aeromonas hydrophila and Vibrio anguillarum. Treatment of cell-free extracts of the LAB strains with proteases revealed the proteinaceous nature of the inhibition. Interestingly, the cell-free extracts containing bacteriocins remained 100% active after treatment up to 100 °C for 30 min or 121 °C for 15 min. Molecular analysis led to identification of the bacteriocins investigated, including enterocins A, B, L50 and P. All of these proteins demonstrated remarkable anti-Listeria activity and were found to be heat-resistant small class IIa bacteriocins. The results presented in this work open the way for potential applications of these LAB strains to the biopreservation of minimally-processed seafood products.