Pierluigi Aldo Di Ciccio

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.

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.

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.

The management of the domestic refrigeration: microbiological status and temperature

Purpose – The purpose of this paper is to provide information on the consumer management of refrigerated food, establish the level and the incidence of bacterial contamination and operating temperatures in domestic refrigerators from north and centre Italy. Design/methodology/approach – A questionnaire was designed involving 24 questions which covered three topics: the socio-demographic data, the domestic management of refrigerated food and general information about the refrigerator. The questionnaire responses were subjected to descriptive statistical analysis and further processed with the multiple correspondence analysis (MCA) and cluster analysis (CA). Based on MCA and CA, 84 refrigerators were selected to assess the temperature and the microbiological status (total viable counts (TVC); Enterobacteriaceae total counts (ETC); Salmonella spp. and Listeria spp.). Findings – Totally, 660 interviews were carried out. The majority of respondents were female (81.4 per cent) and were married (71.4 per cent). ...

Methicillin-resistant food-related Staphylococcus aureus: a review of current knowledge and biofilm formation for future studies and applications.

There is increasing concern about the public health impact of methicillin-resistant Staphylococcus aureus. Food and animal are vectors of transmission, but the contribution of a contaminated environment is not well characterized. With regard to this, staphylococcal biofilms serve as a virulence factor, allowing MRSA strains to adhere to surfaces and other materials used in the food industry. Methicillin resistance and biofilm-forming capacity may contribute to the success of S. aureus as a human pathogen in both health care and community settings and the food production chain. This review summarizes current knowledge about the significance of food- and animal-derived MRSA strains and provides data on attachment and biofilm formation of MRSA. In addition, the impact of quorum sensing on MRSA gene expression and biofilm formation is examined.

Listeria monocytogenes Biofilms in the Wonderland of Food Industry

The foodborne pathogen Listeria monocytogenes is a concern in food safety because of its ability to form biofilm and to persist in food industry. In this mini-review, the issue represented by this pathogen and some of the latest efforts performed in order to investigate the composition of biofilms formed by L. monocytogenes are summarized.

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.

Antimicrobial activity of four essential oils against pigmenting Pseudomonas fluorescens and biofilmproducing Staphylococcus aureus of dairy origin

Essential oils (EOs) are mixtures of secondary metabolites of plant origin with many useful properties, among which the antimicrobial activity is also of interest for the food industry. EOs can exert their antimicrobial potential both directly, in food products and active packaging, and indirectly, as sanitizing and anti-biofilm agents of food facility surfaces. Aim of this research was to evaluate the antimicrobial activity of four EOs (bergamot, cinnamon, manuka and thyme) against Pseudomonas fluorescens and Staphylococcus aureus isolated from milk and dairy products. The chemical composition of EOs was evaluated by Gas Chromatography-Mass Spectrometry analysis. Minimum Inhibitory Concentration values were determined by a microplate method against 9 Ps. fluorescens from marketed mozzarella with blue discoloration defect, and 3 biofilm-producing S. aureus from milk. Reference ATCC strains were included. Pigment production activity by Ps. fluorescens was assessed both in culture and in cheese. EOs of manuka (leptospermone 23%) and thyme (carvacrol 30%, pcymene 20%, thymol 15%) showed the highest antimicrobial activity against S. aureus, MIC values were 0.012%-0.024% and 0.024% v/v, respectively; meanwhile EOs from thyme and cinnamon (cinnamaldehyde 55%) exhibited the best activity against Ps. fluorescens with MIC values of 0.098%-0.195% and 0.195%-0.391% v/v, respectively. The antimicrobial activity of these EOs is promising and they could be exploited in the dairy production chain.

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.