Bacterial diversity on stainless steel surfaces of egg processing companies and potential of selected isolates to spoil liquid whole egg products.

AIMS: To assess the bacterial diversity in the French egg processing industry and to explore the adhesion and spoilage potential of selected bacteria. METHODS AND RESULTS: Sterile stainless steel chips were suspended for 2 months inside the pipelines of seven egg processing companies, before and after the pasteurizer, at warm and cold seasons. After exposure, the bacterial diversity was assessed by 16S rDNA sequencing. The 231 collected isolates were mainly facultative anaerobic Gram positive bacteria, such as Streptococcus, Staphylococcus, Bacillus and Kocuria. Sixty-five representative isolates were further characterized in vitro regarding the potential for adhesion and egg product spoilage. A high diversity was observed from one genus to another. Kocuria and Rothia isolates showed significantly higher adhesion than the isolates of the other genera. Only the isolates belonging to the genera Bacillus and Lysinibacillus, associated with high enzymatic activities on a solid egg-based medium, were able to induce spoilage of liquid whole egg. CONCLUSIONS: Bacteria collected on stainless steel surfaces placed in egg processing industries could be associated to liquid egg product spoilage. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new insights on the bacterial contamination in egg processing companies and represents a first step for the effective control of undesirable bacteria in liquid egg products.

[Pathway to diagnosis and real-life experience of patients believing they are affected by "chronic Lyme disease"]. / Cheminement diagnostique et vécu des patients se pensant atteints de « maladie de Lyme chronique ¼.

OBJECTIVES: Chronic Lyme disease is a subject of scientific and social controversy in both Europe and the United States. The aim of our study was to analyze the pathway to diagnosis of patients believing they were affected by the disease, and to describe their real-life experience. METHOD: A qualitative study was performed with 13 patients declaring themselves to be affected by chronic Lyme disease. Interviews were analyzed by 2 general medical practice interns, supervised by a general practitioner with a diploma in socio-anthropology and an infectious diseases specialist. RESULTS: Internet and other media played a major role in informing the patients or their doctor about the existence and the characteristics of chronic Lyme disease. The diagnosis was confirmed by features considered objective (chronic infection by Borrelia, tick bite, positive serology, beneficial or worsening effects of antibiotics). The long medical diagnosis and treatment process of those interviewed was marked by a conflicted relationship with the medical profession, caused by a feeling of non-recognition and abandonment. They reported their experience as being very painful, both because of the physical pain and also the psychological consequences of their condition. CONCLUSION: Improving the diagnosis and therapeutic management of patients believing themselves to be affected by chronic Lyme disease appears highly necessary both to limit their search for diagnosis and their experience of pain. It could be based on existing guidelines concerning medically unexplained symptoms to which the chronic Lyme disease issue appears quite similar on several points.

SOLEIL shining on the solution-state structure of biomacromolecules by synchrotron X-ray footprinting at the Metrology beamline.

Synchrotron X-ray footprinting complements the techniques commonly used to define the structure of molecules such as crystallography, small-angle X-ray scattering and nuclear magnetic resonance. It is remarkably useful in probing the structure and interactions of proteins with lipids, nucleic acids or with other proteins in solution, often better reflecting the in vivo state dynamics. To date, most X-ray footprinting studies have been carried out at the National Synchrotron Light Source, USA, and at the European Synchrotron Radiation Facility in Grenoble, France. This work presents X-ray footprinting of biomolecules performed for the first time at the X-ray Metrology beamline at the SOLEIL synchrotron radiation source. The installation at this beamline of a stopped-flow apparatus for sample delivery, an irradiation capillary and an automatic sample collector enabled the X-ray footprinting study of the structure of the soluble protein factor H (FH) from the human complement system as well as of the lipid-associated hydrophobic protein S3 oleosin from plant seed. Mass spectrometry analysis showed that the structural integrity of both proteins was not affected by the short exposition to the oxygen radicals produced during the irradiation. Irradiated molecules were subsequently analysed using high-resolution mass spectrometry to identify and locate oxidized amino acids. Moreover, the analyses of FH in its free state and in complex with complement C3b protein have allowed us to create a map of reactive solvent-exposed residues on the surface of FH and to observe the changes in oxidation of FH residues upon C3b binding. Studies of the solvent accessibility of the S3 oleosin show that X-ray footprinting offers also a unique approach to studying the structure of proteins embedded within membranes or lipid bodies. All the biomolecular applications reported herein demonstrate that the Metrology beamline at SOLEIL can be successfully used for synchrotron X-ray footprinting of biomolecules.

Global overview of the risk linked to the Bacillus cereus group in the egg product industry: identification of food safety and food spoilage markers.

AIMS: To evaluate the food safety and spoilage risks associated with psychrotrophic Bacillus cereus group bacteria for the egg product industry and to search for relevant risk markers. METHODS AND RESULTS: A collection of 68 psychrotrophic B. cereus group isolates, coming from pasteurized liquid whole egg products, was analysed through a principal component analysis (PCA) regarding their spoilage and food safety risk potentials. The principal component analysis showed a clear differentiation between two groups within the collection, one half of the isolates representing a safety risk and the other half a spoilage risk. CONCLUSIONS: Relevant risk markers were highlighted by PCA, that is (i) for the food safety risk, the presence of the specific 16S rDNA-1m genetic signature and the ability to grow at 43°C on solid medium and (ii) for the spoilage risk, the presence of the cspA genetic signature. SIGNIFICANCE AND IMPACT OF THE STUDY: This work represents a first step in the development of new diagnostic technologies for the assessment of the microbiological quality of foods likely to be contaminated with psychrotrophic B. cereus group bacteria.

Desorption electrospray ionization mass spectrometry of glycosaminoglycans and their protein noncovalent complex.

Glycosaminoglycans heparin and heparan sulfate are biologically active polysulfated carbohydrates that are among the most challenging biopolymers with regards to their structural analysis and functional assessment. Fragmentation of oligosaccharides and sulfate loss are important hindrance to their analysis by mass spectrometry (MS), requiring thus soft ionization methods. The recently introduced soft ionization method desorption electrospray ionization (DESI) has been applied here to heparin and heparan sulfate oligosaccharides, showing that DESI-MS is well suited for the detection of such fragile biomolecules in their intact form. Characterization of complicated oligosaccharides such as synthetic heparin octadecasulfated dodecasaccharide was successfully achieved. The use of water for a spray solvent instead of denaturing organic solvents allowed the first DESI-MS detection of noncovalent biomolecular complexes between heparin oligosaccharides and the chemokine Stromal Cell-derived Factor-1. The hyphenation of the DESI ion source with the high-resolution LTQ-Orbitrap MS analyzer led to high accuracy of mass measurement and enabled unambiguous determination of the protein-bound sulfated oligosaccharide.

Hyphenation of surface plasmon resonance imaging to matrix-assisted laser desorption ionization mass spectrometry by on-chip mass spectrometry and tandem mass spectrometry analysis.

Most of the recent developments aiming to the coupling between surface plasmon resonance (SPR) and mass spectrometry (MS) are based on the use of a biochip with a limited number of flow cells requiring elution steps for the recovery of the captured biomolecules. In this work, a direct on-chip MALDI-MS detection is presented using a SPRi-sensor biochip in a microarray format that allows a multiplex SPR-MS analysis. The biochip gold surface was functionalized by a self-assembled monolayer (SAM) of short polyoxyethylene (POE) chains carrying a N-hydroxysuccinimide (NHS) group for the immobilization of biomolecules. The SPR measurement of the interaction of grafted antibodies anti-beta-lactoglobulin and anti-ovalbumin with their corresponding antigens indicated that the POE-NHS SAM preserved the binding activity of the antibodies immobilized on the biochips surface. SPR-MS experiments were carried out through MALDI-MS detection of the retained antigens (beta-lactoglobulin and ovalbumin) directly from the biochip surface. Mass spectra were obtained from each distinct spot on the arrayed biochips. Femtomole amounts of specifically retained antigen proteins as determined by SPR were sufficient to obtain good quality mass spectra. These mass spectra showed protein ions corresponding to the specific antigen, without any trace of nonspecific binding. The underivatized portion of the chip was also devoid of nonspecifically bound proteins, indicating that the functionalization of the biochips surface by short polyoxyethylene chains greatly minimizes the unspecific binding. In addition, it allowed on-chip digestion of the specifically bound analyte and coupling with MS/MS experiments, opening numerous applications in the proteomic field.

Effects of liquid phase composition on salt cluster formation in positive ion mode electrospray mass spectrometry: implications for clustering mechanism in electrospray.

Potassium bromate salt clusters, [KBrO3]nKx(x+), formed by electrospray ionization were studied as a function of solution properties. Clusters with up to 4 positive charges were observed. Their abundance, charge state and distribution were shown to vary with the organic solvent in solution. The effects of 7 solvents, including methanol, ethanol, isopropanol, acetonitrile, acetone, pyridine, and 1,4-dioxane, were thoroughly investigated. Solvents with a low dielectric constant and a high viscosity seem to favor clustering in solution but do not systematically allow high charge state ion formation. On the other hand, cluster charge reduction during desolvation was not correlated with solvent cation affinity over the range of solvents examined. However, ion distribution in mass spectra could be rationalized as a combination of these two competing phenomena. Charge state increases with the cluster size but may be reduced during ion desolvation when high cation affinity solvent molecules are actually involved in the ion solvation shell. This assumption could be envisaged in either Iribarne or Dole mechanisms of ion release in the gas phase. However, intensity profiles of multiply charged clusters could only be understood in terms of the ion evaporation mechanism.

Development of irreversible lesions in the brain, heart and kidney following acute and chronic murine malaria infection.

Irreversible pathological lesions were noted in the organs of mice infected with 1 of 3 rodent malaria species: Plasmodium chabaudi chabaudi, P. vinckei petteri and P. yoelii nigeriensis at different times during the course of the primary parasitaemia and long after microscopical clearance of the parasites. Moreover, similar lesions were also obtained when parasite levels were kept below 1% by subcurative drug treatment. The frequency and severity of the lesions correlated with the duration of the infection. Accumulation of tissue damage during chronic low-grade malaria infections has implications for the design of control measures.

Kinetic analysis of the reaction between d(TTGGCCAA) and [Pt(NH3)3(H2O)]2+ by enzymatic degradation of the products and ESI and MALDI mass spectrometries.

The kinetics of the reaction between the octanucleotide d(TTGGCCAA) in the single-stranded form in pure water and the platinum complex [Pt(NH3)3(H2O)]2+ was investigated by electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) mass spectrometries coupled with enzymatic degradation of the adducts. These methods led to the determination of specific rate constants of platination. The global rate constant characteristic of the formation of adducts on each 5'- or 3'-guanine were measured by electrospray ionization analysis. The ratios between the 5'- and 3'-adducts were determined from enzymatic degradation of the final reaction mixture and MALDI analysis. The platination in water is approximately eight times faster than in 0.1 M NaClO4. The selectivity of platination is a factor of 2 in favor of the 5'-guanine, and similar to that observed for the reaction between d(CTGGCTCA) and [Pt(NH3)3(H2O)]2+ in 0.1 M NaClO4.

Neuropeptide Y (NPY) modulatesin vitro gonadotropin in release from rainbow trout pituitary glands.

This work investigated the action of neuropeptide Y (NPY) on thein vitro pituitary release of the maturing gonadotropic hormone (GtH) of the rainbow trout using a perifusion system employing trout balanced salt solution (pH 7.5) at 15°C and a 12.5 ml/h flow rate. In vitellogenic females a 20 minutes NPY application (10(-7) M) induced a 20-30% decrease in GtH secretion. Removal of NPY was followed by a rebound in GTH secretion. On the contrary, in ovulated females, NPY (15 minutes, 10(-7) M) directly stimulated GTH secretion. The greatest stimulation was obtained the day of ovulation where the stimulatory effect of NPY was similar to those induced by s.GnRH in the same conditions, reaching 400% of the basal GTH level. In vitellogenic females treated with 1-4-6 androstadien 3-7 dione, an inhibitor of aromatase activity, the pituitary response to NPY was similar to that obtained in ovulated females. Thus thein vitro action of NPY might depend on thein vivo steroidogenic environment.

Hypothalamic control of prolactin release in the rainbow trout,Salmo gairdneri: in vitro studies.

Hypothalamic control of prolactin (PRL) release in immature rainbow troutSalmo gairdneri was investigated using anin vitro perifusion system of the rostral pars distalis. Hypothalamic extract of trout induced a dose-dependent stimulation of PRL release. A similar effect was observed when infusing the medium from a 24h static incubation of the hypothalamus. Extracts from different control tissues (muscle, liver, gut) did not changein vitro release, thus confirming the specificity of this stimulatory effect. Hypothalamic extract from adult male rat, known to contain PRL release inhibiting factors, stimulatedin vitro PRL secretion in rainbow trout. This suggests that PRL cells are predominantly influenced by PRL releasing factors. Measurement of TRH and serotonin content in trout hypothalamus indicated consistent physiological levels of these two factors. HPLC studies of hypothalamic extract showed that immunoreactive - TRH eluted at the same place as labelled TRH standard. Moreover, pizotifen, a serotonin antagonist, partially inhibited the stimulation observed with trout hypothalamic extract. These results suggest that, in immature rainbow trout, PRL release is under stimulatory hypothalamic control and that serotonin and probably TRH play a major role in this control.

Effect of osmotic pressure on prolactin release in rainbow trout: in vitro studies.

To investigate a possible effect of osmotic pressure on prolactin (PRL) release in rainbow trout, we developed a technique for in vitro perifusion of trout pituitaries. Changes in osmotic pressure similar to those observed in fish plasma during transfer experiments did not induce significant modifications of PRL release. In contrast, high-amplitude variation of osmotic pressure resulted in clear modifications of PRL secretion: hyperosmotic medium caused a reduction in PRL release, while infusion of hyposmotic medium induced a transitory increase in PRL release. By using different concentrations of mannitol, we found that the modifications of prolactin secretion could not be ascribed to alterations of the ionic composition of the medium but actually resulted from variations in the osmotic pressure of the incubation medium. In further experiments osmotic pressure was decreased from 300 to 220 mOsm/kg or from 400 to 300 mOsm/kg; a similar transitory increase in PRL release was observed. Measurement of gonadotropin (GtH) in the perifusion effluent medium showed that PRL and GtH secretion followed similar patterns. Thus, our results suggest a possible mechanical effect of wide changes in osmotic pressure on pituitary cell membranes. These data indicate that the rainbow trout differs notably from nonsalmonid teleost species thus far studied in the lack of sensitivity of its PRL cells to osmotic pressure.

Central GABAergic innervation of the pituitary in goldfish: a radioautographic and immunocytochemical study at the electron microscope level.

The GABAergic innervation of the goldfish pituitary was studied at the light and electron microscope levels by means of radioautography after in vitro incubation in tritiated gamma-aminobutyric acid (GABA) and immunocytochemistry using antibodies against GABA. Following incubation of pituitary fragments in a medium containing tritiated GABA, a selective uptake of the tracer was observed within the digitations of the neurohypophysis. Silver grain clusters were also observed in the adenohypophyseal tissue. At the electron microscope level, this uptake was found to correspond to nerve endings containing small clear and dense-core vesicles. These labeled profiles were located mainly in neurohypophyseal digitations in close apposition with the basement membrane separating the neurohypophysis from the adenohypophysis. However, they were also encountered in direct contact with most adenohypophyseal cell types in the different lobes. These results were confirmed by immunocytochemical data demonstrating the presence of numerous GABA immunoreactive fibers in both anterior and neurointermediate lobes. They were found either in the digitations of the neurohypophysis or in the adenohypophysis in direct contact with the glandular cells with a distribution and an ultrastructural aspect similar to those observed by radioautography. These data demonstrate that the pituitary of teleosts receives a massive GABAergic innervation. Although physiological data providing a functional significance for such an innervation are lacking, the present study suggests that, as already documented in mammals, GABA may be involved in the neuroendocrine regulation of pituitary functions in teleosts.