Domenico Paludi

Characterization of antimicrobial resistance of foodborne Listeria monocytogenes

The objective of this study was to evaluate the susceptibility of 120 Listeria monocytogenes strains isolated from food and food-processing environments to 19 antibiotics currently used in veterinary and human therapy. Susceptibility tests were performed by using the automated VITEK2 system. Apart from penicillin, ampicillin and trimethoprim-sulfamethoxazole, for which clinical breakpoints for Listeria susceptibility testing are defined according to the Clinical and Laboratory Standard Institute (CLSI), in the present study the CLSI criteria for staphylococci were applied. Among the 120 tested strains, 14 (11.7%) displayed resistance to at least one antibiotic. In particular, resistance to one antibiotic was more common than multiple resistance, i.e., 10 (8.3%) isolates were resistant to one antibiotic, 3 (2.5%) to two antibiotics and one (0.8%) to five antibiotics. Resistance to clindamycin was the most common, followed by linezolid, ciprofloxacin, ampicillin and rifampicin, trimethoprim/sulphamethoxazole and, finally, vancomycin and tetracycline. This study shows that L. monocytogenes strains from food and food-processing environments are susceptible to the antibiotics commonly used in veterinary and human listeriosis treatment. Considering that L. monocytogenes is slowly becoming antibiotic resistant, a continued surveillance of emerging antimicrobial resistance of this pathogen is important to ensure effective treatment of human listeriosis. These data are useful in improving background data on antibiotic resistance of strains isolated from food and food environment.

Efficacy of novel acridine derivatives in the inhibition of hPrP90-231 prion protein fragment toxicity.

Quinacrine is one of the few molecules tested to treat patients affected by prion diseases, although the clinical outcome is largely unsatisfactory. To identify novel derivatives with higher neuroprotective activity, we evaluated the effects of a small library of acridine derivatives. The 6-chloro-2-methoxyacridine derivatives bearing on position 9 a quinolizidin-1-ylamino (Q1, Q2) or a quinolizidin-1-ylalkylamino residue (Q3, Q4, Q6, Q7), the thio-bioisoster of Q3 (Q5), the 9-(N-lupinylthiopropyl)amino derivative (Q8) and simple acridines (Q9 and Q10) were considered. We compared the effects of quinacrine and these novel analogues in the inhibition of the cytotoxic activity and protease K (PK) resistance of the human prion protein fragment 90-231 (hPrP90-231). We demonstrate that quinacrine caused a significant reduction of hPrP90-231 toxicity due to its binding to the fragment and the prevention of its conversion in a toxic isoform. All acridine derivatives analyzed showed high affinity binding for hPrP90-231, but only Q3 and Q10, caused a significant reduction of hPrP90-231 cytotoxicity, with higher efficacy than quinacrine. We attempted to correlate the cytoprotective effects of the new compounds with some biochemical parameters (binding affinity to hPrP90-231, intrinsic fluorescence quenching, hydrophobic amino acid exposure), but a direct relationship occurred only with the reduction of PK resistance, likely due to the prevention of the acquisition of the β-sheet-rich toxic conformation. These data represent interesting leads for further modifications of the basic side chain and the substituent pattern of the acridine nucleus to develop novel compounds with improved antiprion activity to be tested in in vivo experimental setting.

Intracellular accumulation of a mild‐denatured monomer of the human PrP fragment 90–231, as possible mechanism of its neurotoxic effects

Because of high tendency of the prion protein (PrP) to aggregate, the exact PrP isoform responsible for prion diseases as well as the pathological mechanism that it activates remains still controversial. In this study, we show that a pre‐fibrillar, monomeric or small oligomeric conformation of the human PrP fragment 90–231 (hPrP90–231), rather than soluble or fibrillar large aggregates, represents the neurotoxic species. In particular, we demonstrate that monomeric mild‐denatured hPrP90–231 (incubated for 1 h at 53°C) induces SH‐SY5Y neuroblastoma cell death, while, when structured in large aggregates, it is ineffective. Using spectroscopic and cellular techniques we demonstrate that this toxic conformer is characterized by a high exposure of hydrophobic regions that favors the intracellular accumulation of the protein. Inside the cells hPrP90–231 is mainly compartmentalized into the lysosomes where it may trigger pro‐apoptotic ‘cell death’ signals. The PrP toxic conformation, which we have obtained inducing a controlled in vitro conformational change of the protein, might mimic mild‐unfolding events occurring in vivo, in the presence of specific mutations, oxidative reactions or proteolysis. Thus, in light of this model, we propose that novel therapeutic strategies, designed to inhibit the interaction of the toxic PrP with the plasmamembrane, could be beneficial to prevent the formation of intracellular neurotoxic aggregates and ultimately the neuronal death.

Characterization of the Proapoptotic Intracellular Mechanisms Induced by a Toxic Conformer of the Recombinant Human Prion Protein Fragment 90–231

Abstract:  Prion diseases comprise a group of fatal neurodegenerative disorders that affect both animals and humans. The transition of the prion protein (PrP) from a mainly α‐structured isoform (PrPC) to a prevalent β‐sheet‐containing protein (PrPSc) is believed to represent a major pathogenetic mechanism in prion diseases. To investigate the linkage between PrP neurotoxicity and its conformation, we used a recombinant prion protein fragment corresponding to the amino acidic sequence 90–231 of human prion protein (hPrP90–231). Using thermal denaturation, we set up an experimental model to induce the process of conversion from PrPC to PrPSc. We report that partial thermal denaturation converts hPrP90–231 into a β‐sheet‐rich isoform, displaying a temperature‐ and time‐dependent conversion into oligomeric structures that share some physico‐chemical characteristics with brain PrPSc. SH‐SY5Y cells were chosen to characterize the potential neurotoxic effect of hPrP90–231 in its different structural conformations. We demonstrated that hPrP90–231 in β‐conformation, but not when α‐structured, powerfully affected the survival of these cells. hPrP90–231 β‐structured caused DNA fragmentation and a significant increase in caspase‐3 proteolytic activity (maximal effects + 170%), suggesting the occurrence of apoptotic cell death. Finally, we investigated the involvement of MAP kinases in the regulation of β‐hPrP90–231‐dependent apoptosis. We observed that the p38 MAP kinase blocker SB203580 prevented the apoptotic cell death evoked by hPrP90–231, and Western blot analysis revealed that the exposure of the cells to the peptide induced p38 phosphorylation. In conclusion, we demonstrate that the hPrP90–231 elicits proapoptotic activity when in β‐sheet‐rich conformation and that this effect is mediated by p38 and caspase‐3 activation.

Different structural stability and toxicity of PrPARR and PrPARQ sheep prion protein variants

The polymorphisms at amino acid residues 136, 154, and 171 in ovine prion protein (PrP) have been associated with different susceptibility to scrapie: animals expressing PrPARQ [PrP(Ala136/Arg154/Gln171)] show vulnerability, whereas those that express PrPARR [PrP(Ala136/Arg154/Arg171)] are resistant to scrapie. The aim of this study was to evaluate the in vitro toxic effects of PrPARR and PrPARQ variants in relation with their structural characteristics. We show that both peptides cause cell death inducing apoptosis but, unexpectedly, the scrapie resistant PrPARR form was more toxic than the scrapie susceptible PrPARQ variant. Moreover, the α‐helical conformation of PrPARR was less stable than that of PrPARQ and the structural determinants responsible of these different conformational stabilities were characterized by spectroscopic analysis. We observed that PrP toxicity was inversely related to protein structural stability, being the unfolded conformation more toxic than the native one. However, the PrPARQ variant displays a higher propensity to form large aggregates than PrPARR. Interestingly, in the presence of small amounts of PrPARR, PrPARQ aggregability was reduced to levels similar to that of PrPARR. Thus, in contrast to PrPARR toxicity, scrapie transmissibility seems to reside in the more stable conformation of PrPARQ that allows the formation of large amyloid fibrils.

The hygiene-sanitary control in the wild game meats

The use of game meat as a food source is currently a growing trend in our country. These products have strong and historic ties with cultural and culinary tradition, but are also appreciated for their sensory and nutritional characteristics. A major contributor to the supply of this type of product is hunting. Practiced since the dawn of time for survival, hunting has evolved into a recreational activity with substantial commercial interests. Of particular importance in this context is hunting of large ungulates. The progressive urbanization of the population has allowed for the re-establishment of bush and wooded areas that represent the ideal habitat of species such as the wild boar, whose numbers are increasing throughout the country. It is therefore clear that implementation of safety rules regarding the hunting and consumption of game meat needs to be urgently addressed. The understanding and application of rules isn’t always easy since the health law is intertwined with that of hunting, and the decision- making power left to the different regions does not contribute to a uniform application throughout the country. The aim of this study was to examine the norms that regulate the use of large wild game meat intended for human consumption and their applicability in hunting activities. From the comparison of the data reported in the literature and our field experience the rules implementation and the problems are evaluated. Operational procedures are then proposed to simplify some of the most difficult aspects and fill in the gaps highlighted.