Whole genome sequencing for typing and characterisation of Listeria monocytogenes isolated in a rabbit meat processing plant

Listeria monocytogenes is a food-borne pathogen able to survive and grow in different environments including food processing plants where it can persist for month or years. In the present study the discriminatory power of Whole Genome Sequencing (WGS)-based analysis (cgMLST) was compared to that of molecular typing methods on 34 L. monocytogenes isolates collected over one year in the same rabbit meat processing plant and belonging to three genotypes (ST14, ST121, ST224). Each genotype included isolates indistinguishable by standard molecular typing methods. The virulence potential of all isolates was assessed by Multi Virulence-Locus Sequence Typing (MVLST) and the investigation of a representative database of virulence determinant genes. The whole genome of each isolate was sequenced on a MiSeq platform. The cgMLST, MVLST, and in silico identification of virulence genes were performed using publicly available tools. Draft genomes included a number of contigs ranging from 13 to 28 and N50 ranging from 456298 to 580604. The coverage ranged from 41 to 187X. The cgMLST showed a significantly superior discriminatory power only in comparison to ribotyping, nevertheless it allows the detection of two singletons belonging to ST14 that were not observed by other molecular methods. All ST14 isolates belonged to VT107, which 7-loci concatenated sequence differs for only 4 nucleotides to VT1 (Epidemic clone III). Analysis of virulence genes showed the presence of a fulllength inlA version in all ST14 isolates and of a mutated version including a premature stop codon (PMSC) associated to attenuated virulence in all ST121 isolates

Characterization and performance of a toluene-degrading biofilm developed on pumice stones

BACKGROUND: Hydrocarbon-degrading biofilms in the treatment of contaminated groundwaters have received increasing attention due to the role played in the so-called "biobarriers". These are bioremediation systems in which a microbial consortium adherent to a solid support is placed across the flow of a contaminated plume, thus promoting biodegradation of the pollutant. RESULTS: A microbial consortium adherent to pumice granules (biofilm) developed from a toluene-enriched microflora in a mini-scale system, following continuous supply of a mineral medium containing toluene, over a 12-month period. Observation by scanning electron microscopy, together with quantification of the biomass attached to pumice, evidenced the presence of abundant exopolymeric material surrounding the cells in the biofilm. Toluene removal monitored during 12-month operation, reached 99%. Identification of the species, based on comparative 16S ribosomal DNA (rDNA) sequence analysis, revealed that Rhodococcus erythropolis and Pseudomonas marginalis were the predominant bacterial species in the microbial consortium. CONCLUSION: A structurally complex toluene-degrading biofilm, mainly formed by Rhodococcus erythropolis and Pseudomonas marginalis, developed on pumice granules, in a mini-scale apparatus continuously fed with toluene

Dissecting the molecular determinants of GABAA receptors current rundown, a hallmark of refractory human epilepsy

GABAA receptors-(Rs) are fundamental for the maintenance of an efficient inhibitory function in the central nervous system (CNS). Their dysfunction is associated with a wide range of CNS disorders, many of which characterized by seizures and epilepsy. Recently, an increased use-dependent desensitization due to a repetitive GABA stimulation (GABAA current rundown) of GABAARs has been associated with drug-resistant temporal lobe epilepsy (TLE). Here, we aimed to investigate the molecular determinants of GABAA current rundown with two different heterologous expression systems (Xenopus oocytes and human embryonic kidney cells; HEK) which allowed us to manipulate receptor stoichiometry and to study the GABAA current rundown on different GABAAR configurations. To this purpose, we performed electrophysiology experiments using two-electrode voltage clamp in oocytes and confirming part of our results in HEK. We found that different degrees of GABAA current rundown can be associated with the expression of different GABAAR beta-subunits reaching the maximum current decrease when functional alpha1beta2 receptors are expressed. Furthermore, the blockade of phosphatases can prevent the current rundown observed in alpha1beta2 GABAARs. Since GABAAR represents one important therapeutic target in the treatment of human epilepsy, our results could open new perspectives on the therapeutic management of drug-resistant patients showing a GABAergic impairment

Burkitt's lymphoma mimicking EBV disease as first sign of vertical HIV infection in an adolescent

Burkitt's Lymphoma (BL) rarely represents the first clinical manifestation of vertical HIV infection in adolescent in Western Europe. We report the case of a 17 year-old boy with two week history of fever and enlarged cervical lymph nodes firstly misdiagnosed as EBV infection, subsequently diagnosed as Burkitt's Lymphoma and vertical HIV infection

Impaired duodenal Palmitoylethanolamide release underlies acid-induced mast cells activation in Functional Dyspepsia

Acid hypersensitivity is claimed to be a symptomatic trigger in functional dyspepsia (FD); however, the neuroimmune pathway(s) and the mediators involved in this process have not been systematically investigated. Palmitoylethanolamide (PEA) is an endogenous compound, able to modulate nociception and inflammation, but its role in FD has never been assessed

SPECIFIC DYSPEPTIC SYMPTOMS ARE ASSOCIATED WITH POOR RESPONSE TO THERAPY IN PATIENTS WITH GASTROESOPHAGEAL REFLUX DISEASE

Background: In gastroesophageal reflux disease (GORD) patients, coexistence of functional dyspepsia (FD) is known to be associated with poor response to proton pump inhibitors (PPIs), but the contribution of specific dyspepsia symptoms has not been systematically investigated yet. Objective: To characterize the impact of dyspepsia symptoms on PPIs response in GORD patients. Methods:. The enrolled subjects were 100 patients with diagnosis of GORD. All patients underwent a 24 hour pH-impedance test, while on PPIs-therapy. Patients were divided into two groups, refractory and responders, according to the persistence of GORD symptoms. A standardized questionnaire for FD was also administered to assess presence of dyspepsia symptoms. Results: In the subgroup of refractory patients FD was more prevalent than in responder ones, with postprandial fullness, nausea, vomiting, early satiation and epigastric pain being significantly prevalent in refractory GORD-patients. In the multivariate analysis only early satiation and vomiting were significantly associated with poor response to PPIs Conclusion: Coexistence of FD is associated with refractory-GORD. We showed that only early satiation and vomiting are risk factors for poor response to PPIs therapy. Our findings suggest that symptoms of early satiation and vomiting would help to identify the subset of PPIs-refractory GORD patients

Development of a High Oleic Cardoon Cell Culture Platform by SAD Overexpression and RNAi-Mediated FAD2.2 Silencing

The development of effective tools for the sustainable supply of phyto-ingredients and natural substances with reduced environmental footprints can help mitigate the dramatic scenario of climate change. Plant cell cultures-based biorefineries can be a technological advancement to face this challenge and offer a potentially unlimited availability of natural substances, in a standardized composition and devoid of the seasonal variability of cultivated plants. Monounsaturated (MUFA) fatty acids are attracting considerable attention as supplements for biodegradable plastics, bio-additives for the cosmetic industry, and bio-lubricants. Cardoon (Cynara cardunculus L. var. altilis) callus cultures accumulate fatty acids and polyphenols and are therefore suitable for large-scale production of biochemicals and valuable compounds, as well as biofuel precursors. With the aim of boosting their potential uses, we designed a biotechnological approach to increase oleic acid content through Agrobacterium tumefaciens-mediated metabolic engineering. Bioinformatic data mining in the C. cardunculus transcriptome allowed the selection and molecular characterization of SAD (stearic acid desaturase) and FAD2.2 (fatty acid desaturase) genes, coding for key enzymes in oleic and linoleic acid formation, as targets for metabolic engineering. A total of 22 and 27 fast-growing independent CcSAD overexpressing (OE) and CcFAD2.2 RNAi knocked out (KO) transgenic lines were obtained. Further characterization of five independent transgenic lines for each construct demonstrated that, successfully, SAD overexpression increased linoleic acid content, e.g., to 42.5%, of the relative fatty acid content, in the CcSADOE6 line compared with 30.4% in the wild type (WT), whereas FAD2.2 silencing reduced linoleic acid in favor of the accumulation of its precursor, oleic acid, e.g., to almost 57% of the relative fatty acid content in the CcFAD2.2KO2 line with respect to 17.7% in the WT. Moreover, CcSADOE6 and CcFAD2.2KO2 were also characterized by a significant increase in total polyphenolic content up to about 4.7 and 4.1 mg/g DW as compared with 2.7 mg/g DW in the WT, mainly due to the accumulation of dicaffeoyl quinic and feruloyl quinic acids. These results pose the basis for the effective creation of an engineered cardoon cells-based biorefinery accumulating high levels of valuable compounds from primary and specialized metabolism to meet the industrial demand for renewable and sustainable sources of innovative bioproducts

Whole genome sequencing based typing and characterisation of Shiga-toxin producing Escherichia coli strains belonging to O157 and O26 serotypes and isolated in dairy farms

In the present study, the genetic relationships as well as the virulome and resistome of newly sequenced O26 and O157 Shiga-toxin producing E. coli (STEC) isolates, collected from dairy farms in Italy, were investigated in comparison to publicly available genomes collected worldwide. The whole genome of Italian isolates was sequenced on Illumina MiSeq Platform. Reads quality control, de novo draft genome assembly, species confirmation and the 7-loci Multi-Locus Sequence Type assignment were performed using INNUca pipeline. Reference-based SNPs calling was performed on O157 and O26 genomes, separately, mapping contigs to high-quality finished genomes. Virulence and antimicrobial resistance determinants were detected in silico using the tool ABRicate. Phylogenetic reconstructions revealed that genomes clustered mainly based on their 7-loci MLST type. The virulome of tested genomes included 190 determinants. O157 genomes carried chu genes associated to heme mediated iron uptake, whereas O26 genomes harboured genes ybt associated to siderophore mediated iron uptake. Resistome analysis showed the presence of tet(34) on all but one O157 genomes and on only one O26 genomes. Only 4 genomes carried genes associated to multiresistance. In the present study, the genes chu and ybt were identified as potential biomarker for the differentiation of O157 and O26 serotypes

Promoter trapping reveals significant differences in integration site selection between MLV and HIV vectors in primary hematopoietic cells

Recent reports have indicated that human immunodeficiency virus (HIV) and murine leukemia virus (MLV) vectors preferentially integrate into active genes. Here, we used a novel approach based on genetic trapping to rapidly score several thousand integration sites and found that MLV vectors trapped cellular promoters more efficiently than HIV vectors. Remarkably, 1 in 5 MLV integrations trapped an active promoter in different cell lines and primary hematopoietic cells. Such frequency was even higher in growth-stimulated lymphocytes. We show that the different behavior of MLV and HIV vectors was dependent on a different integration pattern within transcribed genes. Whereas MLV-based traps showed a strong bias for promoter-proximal integration leading to efficient reporter expression, HIV-based traps integrated throughout transcriptional units and were limited for expression by the distance from the promoter and the reading frame of the targeted gene. Our results indicate a strong propensity of MLV to establish transcriptional interactions with cellular promoters, a behavior that may have evolved to enhance proviral expression and may increase the insertional mutagenesis risk. Promoter trapping efficiency provides a convenient readout to assess transcriptional interactions between the vector and its flanking genes at the integration site and to compare integration site selection among different cell types and in different growth conditions