Molecular typing and genome sequencing allow the identification of persistent Listeria monocytogenes strains and the tracking of the contamination source in food environments.

The presence of Listeria monocytogenes (Lm) in the food processing environment (facilities and products) is a challenging problem in food safety management. Lm is one of the main causes of mortality in foodborne infections, and the trend is continuously increasing. In this study, a collection of 323 Lm strain isolates recovered from food matrices and food industry environments (surfaces and equipment) over four years from 80 food processing facilities was screened using a restriction site-associated tag sequencing (2b-RAD) typing approach developed for Lm. Thirty-six different restriction site-associated DNA (RAD) types (RTs) were identified, most of which correspond to lineage II. RT1, the most represented genotype in our collection and already reported as one of the most prevalent genotypes in the food environment, was significantly associated with meat processing facilities. The sequencing of the genomes of strains belonging to the same RT and isolated in the same facility in different years revealed several clusters of persistence. The definition of the persistent strains (PSs) allowed the identification of the potential source of contamination in the incoming raw meat that is introduced in the facility to be processed. The slaughterhouses, which, according to the European Union (EU) regulation, are not inspected for the presence of Lm could be hotspots for the persistence of Lm PSs.

Rapid detection and quantification of plasmid-mediated colistin resistance genes (mcr-1 to mcr-5) by real-time PCR in bacterial and environmental samples.

AIM: The aim of the study was to validate a rapid method to detect and quantify colistin resistance genes (mcr-1 to mcr-5) by real-time polymerase chain reaction (RT-PCR) in diverse matrices. METHODS AND RESULTS: The detection limit of two newly designed SYBR Green real-time PCR assays for mcr-4 and mcr-5 and of previously published protocols for mcr-1 to mcr-3 was assessed using serial dilutions of reference strains. The assays could detect all five mcr genes with the lower limit of 102 copy numbers. Escherichia coli isolates (n = 1062) and environmental samples (n = 93) were tested for the presence of mcr genes. The assays enabled the detection of colistin resistance genes both in bacterial isolates and in complex environmental samples. CONCLUSIONS: This method represents a set of sensitive, rapid and effective assays for the screening of colistin resistance directly from the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Colistin is an antimicrobial commonly used in animals and has recently emerged as a last-resort treatment in humans. Plasmid-mediated mcr genes confer resistance to colistin and represent a major threat for public health since they can be easily disseminated through horizontal gene transfer. The rapid and sensitive detection of mcr genes is of utmost necessity.

The effects of glyphosate and AMPA on the mediterranean mussel Mytilus galloprovincialis and its microbiota.

Glyphosate, the most widely used herbicide worldwide, targets the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme in the shikimate pathway found in plants and some microorganisms. While the potential for glyphosate to induce a broad range of biological effects in exposed organisms has been demonstrated, the global molecular mechanisms of toxicity and potential effects in bacterial symbionts remain unclear, in particular for ecologically important marine species such as bivalve molluscs. Here, the effects of glyphosate (GLY), its degradation product aminomethylphosphonic acid (AMPA), and a mixture of both (MIX) on the mussel M. galloprovincialis were assessed in a controlled experiment. For the first time, next generation sequencing (RNA-seq and 16S rRNA amplicon sequencing) was used to evaluate such effects at the molecular level in both the host and its respective microbiota. The results suggest that the variable capacity of bacterial species to proliferate in the presence of these compounds and the impairment of host physiological homeostasis due to AMPA and GLY toxicity may cause significant perturbations to the digestive gland microbiota, as well as elicit the spread of potential opportunistic pathogens such as Vibrio spp.. The consequent host-immune system activation identified at the molecular and cellular level could be aimed at controlling changes occurring in the composition of symbiotic microbial communities. Overall, our data raise further concerns about the potential adverse effects of glyphosate and AMPA in marine species, suggesting that both the effects of direct toxicity and the ensuing changes occurring in the host-microbial community must be taken into consideration to determine the overall ecotoxicological hazard of these compounds.

Secukinumab shows high efficacy irrespective of HLA-Cw6 status in patients with moderate-to-severe plaque-type psoriasis: SUPREME study.

BACKGROUND: Understanding genetic variations is important in predicting treatment response and forms the basis for identifying new pharmacogenetic and pharmacogenomic targets for psoriasis treatment. There are limited data on the efficacy of secukinumab in relation to genetic markers. OBJECTIVES: To evaluate the efficacy and safety of secukinumab 300 mg in HLA-Cw6-positive (Cw6-POS) and HLA-Cw6-negative (Cw6-NEG) patients with moderate-to-severe chronic plaque-type psoriasis. METHODS: SUPREME was a 24-week, phase IIIb study with an extension period up to 72 weeks. Primary end point was Psoriasis Area Severity Index (PASI) 90 response rate after 16 weeks. RESULTS: In total, 434 patients were recruited: 185 (42·6%) were Cw6-POS and 246 (56·7%) were Cw6-NEG (three not assessed). Mean ± SD age was 45·2 ± 13·2 years (Cw6-POS 42·7 ± 13·1; Cw6-NEG 47·2 ± 12·9). The baseline PASI score was comparable between the cohorts [Cw6-POS 20·7 ± 8·99; Cw6-NEG 21·5 ± 9·99 (P = 0·777)]. At week 16, PASI 90 was achieved in 80·4% of Cw6-POS and 79·7% of Cw6-NEG patients (difference 0·76; 95% confidence interval -7·04 to 8·23). No differences in absolute PASI at week 16 (Cw6-POS 1·36 ± 3·58; Cw6-NEG 1·18 ± 2·29) were observed. The overall safety profile of secukinumab was consistent with that previously reported. No statistically significant difference was detected in the rate of treatment-emergent adverse events [Cw6-POS 42·7%; Cw6-NEG 49·6% (P = 0·295)]. A high PASI 90 response was achieved with secukinumab with a fast reduction in absolute PASI. CONCLUSIONS: Determination of HLA-Cw6 status for secukinumab therapy is unnecessary, as it is highly effective regardless of HLA-Cw6 status.

Development of a real-time PCR assay for rapid detection and quantification of Photobacterium damselae subsp. piscicida in fish tissues.

The availability of a rapid and accurate method for the diagnosis of Photobacterium damselae subsp. piscicida (Phdp), able to discriminate its strictly correlated subsp. damselae (Phdd), formally known as Vibrio damsela, is essential for managing fish pasteurellosis outbreaks in farmed fish. A single-step, high-sensitivity real-time PCR assay for simultaneous detection and quantification of P. damselae was designed targeting partial of the sequence of the bamB gene and tested for specificity and sensitivity on laboratory-generated samples as well as on experimentally infected seabream tissue samples. With a limit of detection (LOD) of one copy in pure bacterial DNA, the sensitivity was higher than all methods previously reported. Validation in target and non-target bacterial species proved the assay was able to discriminate Phdd-Phdp subspecies from diverse hosts/geographical origins and between non-target species. In addition, two SNPs in the target amplicon region determine two distinctive qPCR dissociation curves distinguishing between Phdp-Phdd. This is the first time that a molecular method for P. damselae diagnosis combines detection, quantification and subspecies identification in one step. The assay holds the potential to improve the knowledge of infection dynamics and the development of better strategies to control an important fish disease.

Microbiota and environmental stress: how pollution affects microbial communities in Manila clams.

Given the crucial role of microbiota in host development, health, and environmental interactions, genomic analyses focusing on host-microbiota interactions should certainly be considered in the investigation of the adaptive mechanisms to environmental stress. Recently, several studies suggested that microbiota associated to digestive tract is a key, although still not fully understood, player that must be considered to assess the toxicity of environmental contaminants. Bacteria-dependent metabolism of xenobiotics may indeed modulate the host toxicity. Conversely, environmental variables (including pollution) may alter the microbial community and/or its metabolic activity leading to host physiological alterations that may contribute to their toxicity. Here, 16s rRNA gene amplicon sequencing has been applied to characterize the hepatopancreas microbiota composition of the Manila clam, Ruditapes philippinarum. The animals were collected in the Venice lagoon area, which is subject to different anthropogenic pressures, mainly represented by the industrial activities of Porto Marghera (PM). Seasonal and geographic differences in clam microbiotas were explored and linked to host response to chemical stress identified in a previous study at the transcriptome level, establishing potential interactions among hosts, microbes, and environmental parameters. The obtained results showed the recurrent presence of putatively detoxifying bacterial taxa in PM clams during winter and over-representation of several metabolic pathways involved in xenobiotic degradation, which suggested the potential for host-microbial synergistic detoxifying actions. Strong interaction between seasonal and chemically-induced responses was also observed, which partially obscured such potentially synergistic actions. Seasonal variables and exposure to toxicants were therefore shown to interact and substantially affect clam microbiota, which appeared to mirror host response to environmental variation. It is clear that understanding how animals respond to chemical stress cannot ignore a key component of such response, the microbiota.

Microbial dynamics during shelf-life of industrial Ricotta cheese and identification of a Bacillus strain as a cause of a pink discolouration.

Dairy products are perishable and have to be preserved from spoilage during the food chain to achieve the desired shelf-life. Ricotta is a typical Italian soft dairy food produced by heat coagulation of whey proteins and is considered to be a light and healthy product. The shelf-life of Ricotta could be extended, as required by the international food trade market; however, heat resistant microflora causes spoilage and poses issues regarding the safety of the product. Next-generation sequencing (NGS) applied to the Ricotta samples defined the composition of the microbial community in-depth during the shelf-life. The analysis demonstrated the predominance of spore-forming bacteria throughout the shelf-life, mostly belonging to Bacillus, Paenibacillus and Clostridium genera. A strain involved in spoilage and causing a pink discolouration of Ricotta was isolated and characterised as Bacillus mycoides/weihenstephanensis. This is the first report of a food discolouration caused by a toxigenic strain belonging to the Bacillus cereus group that resulted the predominant strain in the community of the defective ricotta. These results suggest that the processing of raw materials to eliminate spores and residual microflora could be essential for improving the quality and the safety of the product and to extend the shelf-life of industrial Ricotta.

Fast Thermal Helium Beam diagnostic for measurements of edge electron profiles and fluctuations.

The edge of fusion experiments is a region where strong gradients develop, together with the presence of strong fluctuations due to turbulence. The thermal helium beam diagnostic developed for the RFX-mod experiment allows the measurements with a single diagnostic of both low frequency time evolution of the edge radial profiles of electron density and temperature (tens of hertz), and the high frequency fluctuations (hundreds of kHz). To maximize the collected light, the three HeI lines necessary to be measured for the evaluation of n(e) and T(e) are separated with a spectrograph, and multianode photomultipliers are used as light detectors. The paper describes the diagnostic setup, with the interface hardware with the machine and the optical layout, and the characterization of its performances.

Reprint of 'Tracking the blue: a MLST approach to characterise the Pseudomonas fluorescens group'.

The Pseudomonas fluorescens group comprises several closely related species that are involved in food contamination and spoilage. Specifically, the interest in P. fluorescens as a spoiler of dairy products increased after the cases of "blue mozzarella" that occurred in Italy in 2010. A Multilocus Sequence Typing (MLST) scheme was developed and applied to characterise 136 isolates (reference strains and food borne isolates) at strain level, to reveal the genetic relationships among them and to disclose any possible genetic clustering of phenotypic markers involved in food spoilage (protease, lipase, lecithinase activities and pigmented or fluorescent molecule production). The production of dark blue diffusible pigment was evaluated on several bacterial culture media and directly on mozzarella cheese. The MLST scheme provided precise genotyping at the strain level, and the population analyses of the concatenated sequences allowed major taxa to be defined. This approach was revealed to be suitable for tracking the strains according to their origin, such as dairy plants or food matrices. The genetic analysis revealed the presence of a connection between the blue pigment production and a specific phylogenetic cluster. The development of the online database specific to the P. fluorescens group (http://pubmlst.org/pfluorescens) will facilitate the application of the scheme and the sharing of the data.

Tracking the blue: a MLST approach to characterise the Pseudomonas fluorescens group.

The Pseudomonas fluorescens group comprises several closely related species that are involved in food contamination and spoilage. Specifically, the interest in P. fluorescens as a spoiler of dairy products increased after the cases of "blue mozzarella" that occurred in Italy in 2010. A Multilocus Sequence Typing (MLST) scheme was developed and applied to characterise 136 isolates (reference strains and food borne isolates) at strain level, to reveal the genetic relationships among them and to disclose any possible genetic clustering of phenotypic markers involved in food spoilage (protease, lipase, lecithinase activities and pigmented or fluorescent molecule production). The production of dark blue diffusible pigment was evaluated on several bacterial culture media and directly on mozzarella cheese. The MLST scheme provided precise genotyping at the strain level, and the population analyses of the concatenated sequences allowed major taxa to be defined. This approach was revealed to be suitable for tracking the strains according to their origin, such as dairy plants or food matrices. The genetic analysis revealed the presence of a connection between the blue pigment production and a specific phylogenetic cluster. The development of the online database specific to the P. fluorescens group (http://pubmlst.org/pfluorescens) will facilitate the application of the scheme and the sharing of the data.

Expression profiling of skeletal muscle in young bulls treated with steroidal growth promoters.

Dexamethasone (Dex), alone or in association with estrogens, is often illegally administered per os at very low dosage as a growth promoter in beef cattle, with effects that are opposite to the muscle wasting and atrophy induced by repeated administration at therapeutic dosages. In vitro and in vivo studies have investigated the catabolic effects of Dex at therapeutic doses on skeletal muscle, demonstrating an increase in the expression of GDF8 (myostatin) gene, a well-known negative regulator of skeletal muscle mass, in a dose-dependent way. This suggested a direct role of myostatin in Dex-induced muscle wasting. In the present study, an oligonucleotide microarray platform was used to compare expression profiles of beef cattle muscle in animals treated with either Dex or Dex plus 17-beta estradiol (Estr) administered at subtherapeutic dosage, against untreated controls. Data analysis demonstrates that the expression profiles were strongly affected by Dex treatment with hundreds of genes upregulated with relevant fold-change, whereas seven genes were downregulated including the myostatin gene. On the contrary, the number of differentially regulated genes was lower in response to the addition of Estr to the Dex treatment. Differentially regulated genes were analyzed to describe the effects of these treatments on muscle physiology, highlighting the importance of specific pathways (e.g., Wnt or cytokine signaling) and cellular processes (e.g., cell shape and motility). Finally, the observed differences in the expression profile will allow the development of indirect bio-markers to detect illegal Dex treatments in beef cattle using quantitative RT-PCR.

Ribosomal protein gene-based phylogeny for finer differentiation and classification of phytoplasmas.

Extensive phylogenetic analyses were performed based on sequences of the 16S rRNA gene and two ribosomal protein (rp) genes, rplV (rpl22) and rpsC (rps3), from 46 phytoplasma strains representing 12 phytoplasma 16Sr groups, 16 other mollicutes and 28 Gram-positive walled bacteria. The phylogenetic tree inferred from rp genes had a similar overall topology to that inferred from the 16S rRNA gene. However, the rp gene-based tree gave a more defined phylogenetic interrelationship among mollicutes and Gram-positive walled bacteria. Both phylogenies indicated that mollicutes formed a monophyletic group. Phytoplasmas clustered with Acholeplasma species and formed one clade paraphyletic with a clade consisting of the remaining mollicutes. The closest relatives of mollicutes were low-G+C-content Gram-positive bacteria. Comparative phylogenetic analyses using the 16S rRNA gene and rp genes were performed to evaluate their efficacy in resolving distinct phytoplasma strains. A phylogenetic tree was constructed based on analysis of rp gene sequences from 87 phytoplasma strains belonging to 12 16Sr phytoplasma groups. The phylogenetic relationships among phytoplasmas were generally in agreement with those obtained on the basis of the 16S rRNA gene in the present and previous works. However, the rp gene-based phylogeny allowed for finer resolution of distinct lineages within the phytoplasma 16Sr groups. RFLP analysis of rp gene sequences permitted finer differentiation of phytoplasma strains in a given 16Sr group. In this study, we also designed several semi-universal and 16Sr group-specific rp gene-based primers that allow for the amplification of 11 16Sr group phytoplasmas.

Active-feedback control of the magnetic boundary for magnetohydrodynamic stabilization of a fusion plasma.

Stable operation with control on magnetohydrodynamic modes has been obtained in the modified reversed field experiment employing a set of 192 feedback controlled saddle coils. Improvements of plasma temperature, confinement (twofold), and pulse length (threefold) and, as a consequence of the magnetic fluctuation reduction, strong mitigation of plasma-wall interaction and mode locking are reported.

The potential role of increased adrenal volume in the pathophysiology of obesity-related type 2 diabetes.

The hypothalamic-pituitary-adrenal (HPA) axis seems to play an important role in obesity and Type 2 diabetes (DM). The aim of the present study was to determine the adrenal volume in obese patients with DM in comparison to obese non-diabetic patients. Eleven diabetic obese and 19 non-diabetic obese women were sequentially invited to take part in the study. Computed tomography (CT) scan of the abdomen was performed to determine adrenal volume, visceral (VF) and sc fat (SCF). Daily urinary free cortisol (UFC) was used as a measure of integrated cortisol production. In the diabetic patients, hemoglobin A1c was measured as an index of metabolic control. Compared to nondiabetic controls, patients with diabetes had a significantly higher total adrenal volume (4.29+/-1.50 vs 2.95+/-1.64; p=0.03). A highly significant correlation was detected between VF and VF/SCF ratio and total adrenal volume in the whole group (r=0.36, p=0.04 and r=0.48, p=0.008, respectively). This study, therefore, suggests an association between abdominal obesity, enlarged adrenals and Type 2 diabetes. These findings support the hypothesis that an increased activity of the HPA axis in obese subjects may be involved in the pathogenesis of Type 2 diabetes.

Cytogenetic characterization of Brazilian Paspalum accessions.

Chromosome number, pairing relationship and meiotic behavior were evaluated in 24 Brazilian accessions of different Paspalum species as an initial screening to determine which of them might be useful in an interspecific hybridization program. The analysis showed that six were diploids, 16 tetraploids and two hexaploids. The pairing relationship was typical for the ploidy level and agreed with reported data. However, the meiotic behavior after diakinesis was much more abnormal than expected considering the pairing relationship. There was a high frequency of abnormal tetrads in the majority of accessions.

Experimental and simulated neon spectra in the 10-nm wavelength region from tokamak and reversed field pinch plasmas.

Experimental neon spectra (in the 10-nm region), from the tokamak Tore Supra and the reversed field pinch experiment RFX, have been simulated. The spectra include lines from three neon ionization states, namely Ne(7+), Ne(6+), and Ne(5+) ions. Collisional radiative models have been built for these three Ne ions, considering electron collisional excitation and radiative decay as populating processes of the excited states. These models give photon emission coefficients for the emitted lines at electron density and temperature values corresponding to the experimental situations. Impurity modelling is performed using a one-dimensional impurity transport code, calculating the steady-state radial distribution of the Ne ions. The Ne line brightnesses are evaluated in a post-process subroutine and simulated spectra are obtained. The parts of the spectra corresponding to a single ionization state do not depend on the experimental conditions and show good agreement with the simulated single ionization state spectra. On the other hand, the superposition of the three spectra depends on the experimental conditions, as a consequence of the fact that the ion charge distribution depends not only on the radial profiles of the electron density and temperature, but also of the impurity transport coefficients. Simulations of the Ne spectra (including transport) give confidence in the atomic physics calculations; moreover, they allow the determination of the transport coefficients in the plasma region emitting the considered ionization states.

Oligonucleotides as hybridization probes to localize phytoplasmas in host plants and insect vectors.

ABSTRACT Protocols have been developed using 20- to 24-mer oligodeoxynucleotides, originally designed as polymerase chain reaction primers, as hybridization probes for the nonradioactive detection of Italian clover phyllody (ICPh) phytoplasma in plant (Chrysanthemum carinatum) and leafhopper (Euscelidius variegatus) tissue. In situ hybridization of paraffin-embedded tissue sections was carried out using oligodeoxynucleotides 5' end-labeled with either Cy5 fluorochrome, biotin, or digoxigenin. The Cy5-labeled oligonucleotide probes that hybridized to phytoplasmas present in plant tissue were visualized by confocal microscopy. The biotin- and digoxigeninlabeled probes were detected in both plant and insect tissue using a chromogenic alkaline phosphatase-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate reaction. An enhancement of a signal was observed in plant tissue when a tyramide signal-amplification procedure was incorporated into the biotin or digoxigenin detection systems. The results obtained using these techniques with the ICPh phytoplasma system showed that they can provide a rapid means of confirming vector status in insects. Due to the potential ability of short, labeled, oligonucleotide probes to specifically distinguish between different phytoplasmas present in multiple infections, this technique should provide a powerful new tool for epidemiological and vector ecology studies.

Hemodynamic alterations during massive incisional hernioplasty.

BACKGROUND: The operative treatment of a large abdominal incisional hernia increases intra-abdominal pressure (IAP). This study was done to verify if this IAP elevation acts on the cardiocirculatory function. STUDY DESIGN: Hemodynamic measurements were performed in five patients who underwent massive incisional hernioplasty before and after abdominal wall closure. RESULTS: Reduction of a large abdominal hernia increases (+226 percent) IAP, which can produce serious hemodynamic alterations, manifested in two patients by a fall of cardiac output (-30 percent), without significant variations of heart rate and arterial pressure. Cardiac output is decreased secondary to decreased venous return, despite the increase in measured central venous pressure (CVP) and pulmonary capillary wedge pressure (PCWP). CONCLUSIONS: During massive incisional hernioplasty, CVP cannot be used as an indicator of venous return to the heart as it reflects a composite of venous filling pressure, pleural pressure, and transmitted IAP. Transmural CVP and PCWP, and not directly measured CVP and PCWP, should be used as clinical indicators of venous return to the heart in this situation.

Prevalence of atherosclerotic involvement of the internal carotid artery in hypertensive patients.

The prevalence of atherosclerotic involvement of the internal carotid arteries, as diagnosed through an echo-Doppler imaging system with pulsed Doppler spectral analysis was evaluated in 49 hypertensives who had a negative history for neurological symptoms and 49 matched controls. The prevalence was 24.5% in the hypertensive group and 10.2% in the controls with a statistically significant difference (chi-square = 6.07, P less than 0.01). Two hypertensives had severe stenosis (above 50% diameter reduction) and 7 had potentially embolic lesions (irregular surface, inhomogeneous appearance). No one of the matched controls was as severely involved. We conclude that arterial hypertension can account for enhanced prevalence of carotid artery disease in asymptomatic patients.

Stylar peroxidase and incompatibility reactions in Petunia hybrida.

Self-, cross- and non-pollinated styles of Petunia hybrida were treated with 3-3'-diaminobenzidine for the ultrastructural localization of peroxidase activity. Wall peroxidases were absent in cross-pollinated styles, but they were detectable as an osmiophilic deposit on the cell walls of the outer portion of the transmitting tissue in self- and non-pollinated styles. The cell layer showing peroxidase activity was thicker in the self-pollinated styles than in the non-pollinated ones. In accordance with current hypotheses on the mechanism involved in pollen incompatibility process, it is suggested: that wall peroxidases present in the cells of the outer portion of the transmitting tissue are involved in the gametophytic self-incompatibility of Petunia; that self-pollination causes an increase in the number of cells involved in the rejection process; that non-pollinated styles, which are characterized by the presence of cell wall peroxidases in the outer portion of the transmitting tissue, are 'prepared' to a certain extent for the rejection of incompatible pollen tubes. The removal of peroxidase activity thus seems to be an important step in the compatible pollination process.