Alberto Vergara

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.

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.

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.

Occurrence of Toxoplasma gondii in Carcasses of Pigs Reared in Intensive Systems in Northern Italy

To evaluate the occurrence of Toxoplasma gondii and to genetically characterize its isolates in carcasses of industrial fattening pigs, blood, diaphragm, and heart samples were collected from 375 carcasses of pigs slaughtered to be processed for Parma ham production. Pigs had been bred on approved farms (n = 75) located in the so-called Food Valley in Italy. Sera were examined for immunoglobulin G antibodies to T. gondii by modified agglutination test (MAT). Both heart and diaphragm samples from seropositive carcasses were processed for the presence of T. gondii DNA (B1 locus) by real-time PCR and high resolution melting (HRM) assay. Anti-Toxoplasma antibodies were detected in 2.1% of pig carcasses, with titers from 1:10 to 1:320. T. gondii DNA was detected in all (eight) seropositive carcasses and in 11 (5 heart and 6 diaphragm samples) of 16 samples; that is, it was detected in heart tissue in two subjects, in diaphragm tissue in three subjects, and in both muscle tissues in three subjects. Toxoplasma genotypes were determined in seven of eight carcasses: type III was identified in four carcasses, type II in two, and both III and II in one carcass. The serological findings and the molecular detection of T. gondii strains suggest that cured meat products obtained from industrially bred pigs may be potential sources of toxoplasmosis for humans. Our results provide novel, important information regarding the seroprevalence and molecular prevalence of T. gondii in intensively reared pigs within this specific region of Italy, particularly because Parma ham from this region is known and consumed worldwide. On-farm preventive measures combined with slaughterhouse monitoring of carcasses of pigs bred for cured meat production should never be overlooked to prevent the introduction of T. gondii into the food chain and to ensure safety for consumers of these products.

Microbiological contamination in three large-scale pig slaughterhouses in Northern Italy

The aim of this survey was to obtain data on microbiological contamination of pig carcasses and environments in three large-scale Italian slaughterhouses (identified as A-B-C) located in Northern Italy. Each slaughterhouse was visited six times. Five carcasses and three different sites of the slaughterhouse (before and during slaughter) were sampled on each sampling day. A single pooled caecal sample was taken on each sampling day. A total of 90 carcasses, 108 environmental samples and 18 caecal samples were collected. Samples from pig carcasses and slaughterhouse environment were analyzed for total viable count (TVC), Enterobacteriaceae count (EBC) and Salmonella. The caecal contents were examined for Salmonella. Carcasses from slaughterhouse A presented the greatest TVC and EBC mean log value, whereas environmental samples collected during slaughter activities from slaughterhouse C showed the greatest TVC and EBC mean log value. As far as the environmental samples collected before slaughter activities are concerned, an average up to 6 log10 colony forming unit (CFU)/cm2 TVC in two slaughter plants (A and C) and 5 log10 CFU/cm2 TVC in one slaughter plant (B) was detected. Salmonella was recovered in two slaughterhouses (A and B). Four different Salmonella serotypes were detected in the positive samples (11). Within serotype S. Rissen and S. Typhimurium monophasic-variant isolates, two pulsed-field gel electrophoresis patterns were identified. The findings in this survey suggest that carcass contamination is influenced by the slaughterhouse plant and this could be a result of differences in line speed. The results of environmental sampling have not shown an association with the slaughterhouse plant.

Epidemiological survey on cystic echinococcosis in wild boar from Central Italy

Despite the wide distribution of wild boar populations in Italy and the increase of its diffusion in urbanized areas, only one case report has described the occurrence of Echinococcus granulosus s.l. in a wild boar from Marche (Central Italy). The present study investigated the presence of E. granulosus sensu lato with an epidemiological survey on wild boars from central Italy that had been killed during hunting season. Seven hundred sixty-five (765) adult wild boars were examined during the 2016–2017 hunting season. Of these animals, 1.0% (8/765) were positive to E. granulosus s.l. with a fertility of 0.3% (2/765), and 2.9% animals (22/765) were positive for the metacestode stage of Taenia hydatigena (Cysticercus tenuicollis), while 0.5% (4/765) showed mixed infection (E. granulosus s.l. + T. hydatigena). Sixteen hydatids were found, of which 12.5% were fertile, 37.5% were sterile, 31.3% were caseous, and 18.8% were calcified. Eight hydatids (two fertile and six sterile cysts) were molecularly characterized by analysis of the mitochondrial gene, cytochrome c oxidase subunit 1 (cox1), and the NADH dehydrogenase subunit 1 (ND1). Hydatids found in wild boars were characterized as E. granulosus sensu stricto (G1 genotype). The present survey represents the first epidemiological study on cystic echinococcosis in wild boar in Italy which highlights the need for more extensive epidemiological investigations to determine the causal factors, economic impact, and public health importance of the disease in this livestock-wildlife setting.

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.

SURVEY ON SOLIDARITY GROUPS OF BUYERS: CRITICAL ISSUES

This work is intended to provide, through the analysis of the dynamics of purchase of solidarity groups of buyers, a contribution to the study of current trends that are affecting the alternative to the dominant agro-food industry. Through the use of a questionnaire the paper investigated the perception as consumers this particular class of buyers have of themselves and how their concepts of “conscious choice” and “responsibility” drive their shopping list and consequently influence the growth and development of the Short Supply Chain, as the work remarked the progressive and notable increment of commerce and families circling around this young but widespreading phenomenon. Particularly striking, in fact, emerge from the role that consumers are becoming part of this phenomenon and the most of them have matured during time the idea that biological and hand-made products are for themselves also Safe, while the Long Supply Chain should incarnate the role of an alienating and wholly food system. The analysis of the questionnaires revealed some problems concerning the methods of transport and storage.