A. Ianieri

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.

Mineral composition of Italian salami and effect of NaCl partial replacement on compositional, physico-chemical and sensory parameters

In 2008 the European Commission developed an EU framework for dietary sodium chloride (NaCl) reduction in order to achieve the World Health Organization recommendations for no more than 5 g/day/person. This initiative is based on four elements: investigate the national data available on NaCl consumption and current NaCl levels of foods, develop actions to raise public awareness, develop reformulation actions with industry/catering, monitor and evaluate actions and reformulations. The initiative is working towards a reduction in NaCl of 16% over 4 years against the 2008 levels and is concentrated on meat products, bread, cheese, and ready meals. In this context, NaCl content and mineral composition of commercial Italian salami were investigated to provide information on their current mineral levels. Moreover, a technological intervention based on NaCl partial replacement by other chloride salts was investigated on Cacciatore salami, a typical Italian dry fermented sausage, as a strategy to decrease the sodium (Na) content of cured meat products. The effect of NaCl partial replacement by KCl, MgCl2, and CaCl2 in some compositional, physicochemical, and sensory properties of Cacciatore salami was evaluated. A 50% reduction of NaCl used for salami manufacture and its replacement by a mixture of KCl, CaCl2, and MgCl2 allowed a 40% lowering of Na content with limited detrimental effects on sensory attributes. Although no effects were observed on pH, water activity, proximate, and free fatty acid composition in reduced sodium Cacciatore salami formulation compared to the traditional one, the NaCl partial replacement induced a significant increase of lipid oxidation.

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.

Longitudinal study on the sources of Listeria monocytogenes contamination in cold-smoked salmon and its processing environment in Italy

The aim of the present study was to investigate the sources of Listeria monocytogenes contamination in a cold smoked salmon processing environment over a period of six years (2003-2008). A total of 170 samples of raw material, semi-processed, final product and processing surfaces at different production stages were tested for the presence of L. monocytogenes. The L. monocytogenes isolates were characterized by multiplex PCR for the analysis of virulence factors and for serogrouping. The routes of contamination over the six year period were traced by PFGE. L. monocytogenes was isolated from 24% of the raw salmon samples, 14% of the semi-processed products and 12% of the final products. Among the environmental samples, 16% were positive for L. monocytogenes. Serotyping yielded three serovars: 1/2a, 1/2b, 4b, with the majority belonging to serovars 1/2a (46%) and 1/2b (39%). PFGE yielded 14 profiles: two of them were repeatedly isolated in 2005-2006 and in 2007-2008 mainly from the processing environment and final products but also from raw materials. The results of this longitudinal study highlighted that contamination of smoked salmon occurs mainly during processing rather than originating from raw materials, even if raw fish can be a contamination source of the working environment. Molecular subtyping is critical for the identification of the contamination routes of L. monocytogenes and its niches into the production plant when control strategies must be implemented with the aim to reduce its prevalence during manufacturing.

Detection of irradiated beef by nuclear magnetic resonance lipid profiling combined with chemometric techniques

The combination of (1)H NMR lipid profiling with multivariate analysis was applied to differentiate irradiated and non-irradiated beef. Two pattern recognition chemometric procedures, stepwise linear discriminant analysis (sLDA) and artificial neural networks (ANNs), provided a successful discrimination between the groups investigated. sLDA allowed the classification of 100% of the samples into irradiated or non-irradiated beef groups; the same result was obtained by ANNs using the 1 kGy irradiation dose as discriminant value suggested by the network. Furthermore, sLDA allowed the classification of 81.9% of the beef samples according to the irradiation dose (0, 2.5, 4.5 and 8 kGy). (1)H NMR lipid profiling, coupled with multivariate analysis may be considered a suitable and promising screening tool for the rapid detection of irradiated meat in official control of food.

Polymorphism of actA gene is not related to in vitro virulence of Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen which is able to cause serious disease both in humans and in animals. Several studies have demonstrated variations in the levels of virulence among L. monocytogenes strains. Invasion and growth ability of L. monocytogenes into cultured cells have been used to evaluate its pathogenicity. In particular, invasiveness and growth ability have been typically investigated using HeLa cell line. This study aimed to provide further insights on the virulence potential as well as on the molecular and phenotypic characteristics of L. monocytogenes isolated both from food sources and food environments. Thirty-eight isolates were tested for cell invasion and intracellular growth. Among the latter, 15 strains exhibited a high invasion index (I.I.); 18 strains showed intermediate II and 5 isolates revealed a low II. Regarding intracellular growth, all tested isolates had a replication time between 2 and 6h. Furthermore, nine virulence-associated genes (hlyA, actA, inlA, inlB, iap, plcA, plcB, mpl, prfA) were investigated by the multiplex PCR assay. All tested virulence genes were detected in all strains. Interestingly, a polymorphism was observed in the actA gene. However, the polymorphism could not be related to a different level of invasion or intracellular growth. In conclusion, data presented in this study have revealed considerable differences in the ability of L. monocytogenes strains to invade host cells and suggest the presence of additional factors that may contribute to adhesion and invasion. Virulence of L. monocytogenes is still not fully understood in some respects. Further studies focused on the mechanisms of L. monocytogenes pathogenicity together with the development of more reliable and efficient methods for virulence determination in this species are still required.

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.

Metabolic profiling by 1H NMR of ground beef irradiated at different irradiation doses

This work describes a metabolic profiling study of non-irradiated and irradiated beef (at 2.5, 4.5 and 8 kGy) using (1)H NMR and chemometrics. The assignment of all major NMR signals of the aqueous/methanolic extracts was performed. A comprehensive multivariate data analysis proved the ability to distinguish between the irradiated and non-irradiated beef. Classification trees revealed that three metabolites (glycerol, lactic acid esters and tyramine or a p-substituted phenolic compound) are important biomarkers for classification of the irradiated and non-irradiated beef samples. Overall, the achieved metabolomic results show that the changes in the metabolic profile of meat provide a valuable insight to be used in detecting irradiated beef. The use of the NMR-based approach simplifies sample preparation and decrease the time required for analysis, compared to available official analytical procedures.

Polycyclic Aromatic Hydrocarbons in Fresh and Cold-Smoked Atlantic Salmon Fillets

The occurrence of polycyclic aromatic hydrocarbons (PAHs) in smoked fish as a consequence of cold smoking was studied. Raw fillets of Salmo salar from Norway or the Irish Sea were sampled in a modern smokehouse and examined for PAH content. The same fillets, labeled with an identification number, were sampled immediately after the smoking process and analyzed. Among the investigated compounds, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, and benzo[ghi]perylene were detected in both raw and smoked fillets. No significant difference (P < 0.01) was observed between raw and smoked samples in the concentrations of six PAHs, but significant differences were found for fluorene, anthracene, fluoranthene, benz[a]anthracene, and benzo[ghi]perylene. Results confirm that PAHs concentrations in smoked fish are the product of both sea pollution and the smoking process. A modern smoking plant with an external smoke generator and a mild treatment as described here will not add significantly to the concentration of PAHs, except for some compounds.

A Look inside the Listeria monocytogenes Biofilms Extracellular Matrix

Listeria monocytogenes is a foodborne pathogen able to persist in food industry and is responsible for a severe illness called listeriosis. The ability of L. monocytogenes to persist in environments is due to its capacity to form biofilms that are a sessile community of microorganisms embedded in a self-produced matrix of extracellular polymeric substances (EPS’s). In this review, we summarized recent efforts performed in order to better characterize the polymeric substances that compose the extracellular matrix (ECM) of L. monocytogenes biofilms. EPS extraction and analysis led to the identification of polysaccharides, proteins, extracellular DNA, and other molecules within the listerial ECM. All this knowledge will be useful for increasing food protection, suggesting effective strategies for the minimization of persistence of L. monocytogenes in food industry environments.