Antimicrobial resistance pattern of methicillin-resistant Staphylococcus aureus in the food industry.

There is increasing concern about the impact on public health of methicillin-resistant Staphylococcus aureus (MRSA) associated with animal food products. MRSA remains a serious problem because of the high incidence and multidrug resistance of the strains, even for strains isolated from foods, food environments and food handlers. The objectives of this study are: (i) to evaluate the susceptibility of S. aureus strains isolated from food, food handlers and food-processing environments to 14 antibiotics currently used in veterinary and human therapy; (ii) to assess the presence of the mecA gene. A total of 1007 samples were collected from food, food handlers, and environments and were analyzed for the presence of S. aureus. S. aureus was present in 165 of the 1007 samples. A total of 157 isolates were methicillin-susceptible S. aureus (MSSA) and 8 isolates were MRSA. In particular, out of 8 MRSA strains detected, 4 strains harboured the mecA gene. All MRSA strains were resistant to at least one of the tested antibiotics and 6 strains demonstrated multi-resistance. Considering the high level of resistances in S. aureus and the isolation of MRSA strains, the surveillance of antimicrobial resistance and the spreading of this pathogen is of crucial importance in the food production chain. These data are useful in improving background data on antimicrobial resistance of S. aureus isolated from food, processing environments and food handlers, supporting the prudent use of antibiotics and the development of international control programs.

Influence of temperature on biofilm formation by Listeria monocytogenes on various food-contact surfaces: relationship with motility and cell surface hydrophobicity.

AIMS: To assess the ability of Listeria monocytogenes to form biofilm on different food-contact surfaces with regard to different temperatures, cellular hydrophobicity and motility. METHODS AND RESULTS: Forty-four L. monocytogenes strains from food and food environment were tested for biofilm formation by crystal violet staining. Biofilm levels were significantly higher on glass at 4, 12 and 22 degrees C, as compared with polystyrene and stainless steel. At 37 degrees C, L. monocytogenes produced biofilm at significantly higher levels on glass and stainless steel, as compared with polystyrene. Hydrophobicity was significantly (P < 0.05) higher at 37 degrees C than at 4, 12 and 22 degrees C. Thirty (68.2%) of 44 strains tested showed swimming at 22 degrees C and 4 (9.1%) of those were also motile at 12 degrees C. No correlation was observed between swimming and biofilm production. CONCLUSIONS: L. monocytogenes can adhere to and form biofilms on food-processing surfaces. Biofilm formation is significantly influenced by temperature, probably modifying cell surface hydrophobicity. SIGNIFICANCE AND IMPACTS OF THE STUDY: Biofilm formation creates major problems in the food industry because it may represent an important source of food contamination. Our results are therefore important in finding ways to prevent contamination because they contribute to a better understanding on how L. monocytogenes can establish biofilms in food industry and therefore survive in the processing environment.

Conformation dependent pro-apoptotic activity of the recombinant human prion protein fragment 90-231.

The transition of prion protein from a mainly alpha-structured isoform (PrPC) to a beta sheet-containing protein (PrPSc) represents a major pathogenetic mechanism in prion diseases. To study the role of PrP structural conformation in prion-dependent neurodegeneration, we analysed the neurotoxicity of PrP in alpha and beta conformations, using a recombinant protein encompassing amino acids 90-231 of the human PrP (hPrP90-231). Using controlled thermal denaturation (53 degrees C, 1h) we converted hPrP90-231 in a structural isoform displaying PrPSc-related characteristics: high beta sheet content, increased aggregability and a slight increase in the resistance to protease K. In virtue of these structural changes, hPrP90-231 powerfully affected the survival of SH-SY5Y cells, inducing a caspase-3 and p38- dependent apoptosis. Conversely, in the native alpha-helix-rich conformation, hPrP90-231 did not show significant cell toxicity. The relationship between the structural state of hPrP90-231 and its neurotoxicity was demonstrated, inducing the thermal denaturation of the peptide in the presence of Congo red that prevented both the transition of hPrP90-231 into a beta-rich isoform and the acquisition of toxic properties. In conclusion, we report that the toxicity of hPrP90-231 is dependent on its three-dimensional structure, as is supposed to occur for the pathogen PrP during TSE.

Identification of a conserved N-capping box important for the structural autonomy of the prion alpha 3-helix: the disease associated D202N mutation destabilizes the helical conformation.

Peptides corresponding to three alpha helices present in the C-terminal region of the human prion protein have been synthesized and their structural autonomy analyzed by circular dichroism (CD) and NMR spectroscopy. The results obtained indicate that the protein fragment corresponding to the alpha 3-helix, in contrast to alpha 1 and alpha 2 peptides, shows a complete structural autonomy. The chemical shifts values found for NH and CHalpha resonance of the isolated alpha 3 peptide, formed by 30 aminoacid residues, were markedly and surprisingly similar to the corresponding values of the alpha 3-helix in the protein. The structural autonomy of the alpha 3-helix is profoundly determined by the presence of the conserved capping box and, in part, by the ionic bond formed between Glu200 and Lys204. On the basis of these observations a novel PrP consensus pattern, centered on the alpha 3-helix region, has been defined. The data indicate that this autonomous and highly conserved region of the PrPc likely plays a critical role in folding and stability. This gives an explanation of why many of pathogenic mutations occur in this part of the molecule, sharing relevant effects on the overall protein conformation. In particular the D202N capping mutation almost completely destabilizes the isolated alpha 3 peptide. While it is well known that the D202N substitution is associated with a GSS disease, the possible structural basis of this fatal pathology has never been investigated. We propose that a lower alpha 3-helical propensity leading to a major destabilization of the PrPc molecule initiates the pathogenic process associated with D202N capping mutation.

Chemokines as markers for parasite-induced inflammation and tumors.

Chemokines are a group of small secreted proteins (8-10 kDa) produced and released by a wide variety of cell types. They were originally described as mediators of leukocyte recruitment, which is essential in acute and chronic inflammation. They also play a critical role in many pathophysiological processes such as allergic responses, infections and autoimmune diseases, tumor growth and hematopoietic development. This review introduces the three supergene families of chemokines (CXC, CC and C) with emphasis on their important role in different states in humans and in animal models with parasitic diseases. The concentration of transcription and translation of the cytokines and chemokines in the parasitic diseases may be an important marker for evaluation of the inflammatory state.

Purification and characterisation of two GST's forms from Rhizobium leguminosarum with a high affinity to herbicides.

Cytosolic glutathione transferases are a family of multifunctional proteins that catalyse the conjugation of GSH to a large variety of endogenous and exogenous compounds. These enzymes have been widely studied in mammals and, to a lesser extent, in plants. In plants, GSTs can detoxify herbicides; they are also induced by pathogenic infection and are likely to be involved in defence responses. GSTs are found in pathogenic and not pathogenic prokaryotes but the functional role played by these enzymes in the cell still remains to be clarified. Here we report the purification and characterisation of two GST forms from Rhizobium leguminosarum that play a very important role in agriculture by inducing nitrogen-fixing nodules on the roots of legumes. These bacterial GSTs from R. leguminosarum have immunological characteristics that are different among them and they are characterised both by a high affinity to herbicides.

Prion protein fragment 106-126 induces a p38 MAP kinase-dependent apoptosis in SH-SY5Y neuroblastoma cells independently from the amyloid fibril formation.

Prion diseases are neurodegenerative disorders of the central nervous system of humans and animals, characterized by spongiform degeneration of the central nervous system, astrogliosis, and deposition of amyloid into the brain. The conversion of a cellular glycoprotein (prion protein, PrP(C)) into an altered isoform (PrP(Sc)) has been proposed to represent the causative event responsible for these diseases. The peptide corresponding to the residues 106-126 of PrP sequence (PrP106-126) is largely used to explore the neurotoxic mechanisms underlying the prion diseases. We investigated the intracellular signaling responsible for PrP106-126-dependent cell death in the SH-SY5Y human neuroblastoma cell line. In these cells, PrP106-126 treatment induced apoptotic cell death and the activation of caspase-3. The p38 MAP-kinase blockers (SB203580 and PD169316) prevented the apoptotic cell death evoked by PrP106-126 and Western blot analysis revealed that the exposure of the cells to the peptide induced p38 activation. However, whether the neuronal toxicity of PrP106-126 is caused by a soluble or fibrillar form of this peptide is still unknown. In this study, we correlated the structural state of this peptide with its neurotoxicity. We show that the two conserved glycines in position 114 and 119 prevent the peptide to assume a structured conformation, favoring its aggregation in amyloid fibrils. The substitution of both glycines with alanine residues (PrP106-126AA) generates a soluble nonamyloidogenic peptide, that retained its toxic properties when incubated with neuroblastoma cells. These data show that the amyloid aggregation is not necessary for the induction of the toxic effects of PrP106-126.

Expression in E. coli and purification of recombinant fragments of wild type and mutant human prion protein.

Prion diseases are fatal neurodegenerative disorders of the CNS of men and animals, characterized by spongiform degeneration of the CNS, astrogliosis and deposition of amyloid into the brain. The conversion of a cellular glycoprotein (the prion protein, PrP(C)) into an altered isoform (the prion scrapie, PrP(Sc)), which accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism, is currently believed to represent the etiologic agent responsible for these diseases. Synthetic or recombinant polypeptides are commonly used to elucidate the mechanism of proteins involved in neurodegenerative diseases. Here we describe a procedure, which allows the synthesis and purification in its native folding, of the human prion protein fragment 90-231, corresponding to the protease resistant core of PrP(Sc). We synthesized the polypeptides 90-231 of both the wild type and the E200K mutant isoforms of PrP. Using a gluthatione S-transferase (GST) fusion protein approach, milligram amounts of polypeptides were obtained after expression in E. coli. The recovery of the purified fusion protein was monitored following the evaluation of the GST activity. The PrP fragment was released from the fusion protein immobilized on a glutathione-coupled agarose resin by direct cleavage with thrombin. The recombinant protein was identified by comassie stained acrylamide gel and by immunoblotting employing a monoclonal anti-PrP antibody. The peptide purified by gel filtration chromatography showed mainly an alpha-helix structure, as analysed by circular dichroism (CD) and an intact disulfide bridge. The same procedure was also successfully employed to synthesize and purify the E200K mutant PrP fragment.

The folding and stability of human alpha class glutathione transferase A1-1 depend on distinct roles of a conserved N-capping box and hydrophobic staple motif.

An N-capping box and a hydrophobic staple motif are strictly conserved in the core of all known glutathione S-transferases (GST). In the present work, mutations of hGSTA1-1 enzyme residues forming these motifs have been generated. The analysis of S154A, D157A, and S154A/D157A capping mutants indicate that the removal of this local signal destabilizes the protein. The fact that the third helical residue D157A mutation (N-3) was much more destabilizing than the first helical residue S154A mutation (N-cap) suggests that the appropriate conformation of the conserved substructure formed by the alpha 6-helix and preceding loop (GST motif II) is crucial for the overall protein stability. The refolding study of GSTA1-1 variants supports the prediction that this subdomain could represent a nucleation site of refolding. The analysis of L153A, I158A, L153G, and L153A/I158A hydrophobic staple mutants indicate that the removal of this motif destabilizes the GSTA1-1 structure as well as its refolding transition state. The hydrophobic staple interaction favors essential inter-domain contacts and, thereby, in contrast to capping interactions, accelerates the enzyme reactivation. Its strict conservation in the GST system supports the suggestion that this local signal could represent an evolutionarily conserved determinant for rapid folding.

Morphological and biochemical variations of Haemophilus influenzae type b induced by pH and temperature changes.

Haemophilus influenzae type b ATCC 10211 was cultured at different temperatures (25 degrees C-49 degrees C) and pH values (5.7-8.7) either in liquid or semisolid medium. Morphological variations of individual cells were noted by optical microscopy depending upon the conditions of growth. At higher temperatures filaments were produced whereby the length of individual cells increased compared to cultures grown at 37 degrees C. Filaments were also observed at lower pH values. Culture conditions also affected colonial morphology. At low pH values colonies had an enhanced lobulated contour and were more wrinkly and rougher than at higher pH. The changes in cellular and colonial morphology were correlated with distinct outer membrane protein profiles. The changes in temperature and pH did not affect identification of the microorganism by the API system.

Conformational properties of five peptides corresponding to the entire sequence of glutathione transferase domain II.

Five peptides matching the helices alpha4, alpha5, alpha6, alpha7, and alpha8, spanning the entire sequence of domain II of pG-STP1-1, have been synthesized and their conformations analyzed by far-UV CD spectroscopy. The results show that a5, a7, and a8 peptides are unstructured in water/2,2,2-trifluoroethanol (TFE) solutions. The a4-peptide also adopts random conformations in aqueous solvent. Moreover, the relative low helical content (20%), estimated for this peptide in the presence of 30% (v/v) TFE, suggests that the sequence of this protein fragment does not possess sufficient information for a strong helical propensity. On the contrary, the synthesized a6 peptide, in the presence of TFE, showed a relevant structural autonomy with a helical content (41%) which was significantly higher than that estimated, under the same conditions, for all other peptides. More in general in the presence of solvents less polar than water, the isolated a6 peptide shows the same helical conformation adopted by the corresponding alpha6-helix in the hydrophobic core of the protein. A n-capping box motif, strictly conserved at the N-terminal of the alpha6-helix of all GST and related protein including eucaryotic translation elongation factor (EF1gamma) and the yeast prion protein Ure2, plays an important role in the alpha-helix nucleation and stability of this protein fragment. The results suggest that the alpha6-helix might represent a nucleation site of GST folding and that the helical conformation of this region of the protein is an important requirement during earlier events of GST refolding.

Leishmania antibodies in children’s serum samples in correlation with the disease in dogs.

Sera of 489 children from Northern Greece aged between 6 months and 15 years of age and aflicted with different clinical entities, were tested for anti – Leishmania infantum specific IgG and IgM antibodies, using an ELISA (enzyme linked immunosorbent assay) technique. In this survey, a remarkably high percentage (8.5%) of hospitalized children reacted positively to this method. Twenty three out of 489 children (4.7%) had IgG antibodies, seventeen (3.5%) IgM, while two (0.4%) had both IgG and IgM antibodies against soluble antigen of L. infantum promastigotes. Females had a higher seropositivity than males. The highest prevalence was observed in males aged between 6 months and 5 years old (10 out of 19), while the lowest was observed also in males aged between 11 and 15 years old (5 out of 11). Seropositivity rate was higher in children below 5 years of age. Some epidemiologic, as well as clinical data of canine Leishmaniosis from Northern Greece are discussed.

Virulence factors of salmonella ser. enteritidis strains isolated in Italy from food-borne outbreaks.

Twelve Salmonella ser. Enteritidis strains phage type 4 isolated in Italy from different food-borne outbreaks were characterized for the expression of different virulence traits, for antibiotic resistance, and for plasmid DNA profile. All the twelve S. Enteritidis strains were able to invade and multiply within HeLa cell monolayers, even if at a lower efficiency if compared to an invasive Shigella flexneri strain. The strains were not hemolytic and produced only a moderate-level cytotoxic effect on HeLa cell monolayers. Moreover, all the strains examined produced mannose-sensitive hemagglutination with chicken erythrocytes but were not able to adhere to tissue culture cells. The strains did not produce the hydroxamate-type siderophore aerobactin or the specific ferric-aerobactin receptor. The S. Enteritidis strains were resistant only to spectinomycin, and eleven strains harbored a 38 MDa non-conjugative plasmid, while one strain harbored a 64 MDa conjugative plasmid which carried a colicinogenic activity-encoding locus. The uniformity of antibiotic resistance pattern, of the plasmid DNA content, and of the virulence factors produced indicated that the S. Enteritidis clinical isolates examined are clonally-related.

SV40 expression in human neoplastic and non-neoplastic tissues: perspectives on diagnosis, prognosis and therapy of human malignant mesothelioma.

We have recently demonstrated the association of SV40 and human pleural malignant mesothelioma. Here, we have investigated whether SV40 viral sequences may be associated with other human tumours or other non-neoplastic pathology and whether SV40 DNA or protein expression may be of diagnostic, prognostic or therapeutic relevance. DNA was extracted from paraffin embedded tissues. SV40, JC and BK viral sequences were detected by the polymerase chain reaction and molecular hybridization with specific probes. The screening with three different sets of SV40-related primers demonstrated that 7/18 (38.8%) mesothelioma specimens were SV40 positive as well as 5/18 (27.7%) tubercular pleural lesions. None of the 18 lung cancers, nor the 20 pleural non-specific inflammatory specimens tested were positive. Twenty-five blood samples and 18 urinary sediments from MM patients were also negative. We have also found that SV40 Tag proteins are present in mesothelioma cells and tumours. Tag proteins may interfere with tumour suppressor gene products, such as p53. Preliminary results suggest that wild type p53 transgene expression, obtained after infection with recombinant adenovirus (AdCMV.p53), inhibited in vitro and in vivo proliferation, inducing apoptosis of mesothelioma cells. Infections with control viruses were ineffective. Thus, SV40 DNA and Tag expression in mesothelioma tumour cells, though probably not relevant for diagnostic or prognostic purposes, may be crucial for innovative gene therapy strategies.