Domenico Meloni

Listeria monocytogenes in RTE foods marketed in Italy: Prevalence and automated EcoRI ribotyping of the isolates

The aims of the present study were: (a) to investigate the prevalence and the enumeration of Listeria monocytogenes in 200 samples of ready to eat (RTE) foods of animal and vegetal origin collected from different outlets and processing plants in Sardinia; (b) to characterize the isolates by phenotypical and molecular methods; (c) to analyze a subset of 42 L. monocytogenes by automated EcoRI ribotyping in order to predict the strains potential virulence for humans. The strains were isolated from: smoked fish products, cooked marinated products, meat products and pre-packaged mixed vegetable salads. Of the samples tested, 22% were positive for Listeria spp. The prevalence of L. monocytogenes was 9.5%, while the level of L. monocytogenes in the positive samples was <10 cfu/g in 94.7% of cases. EcoRI ribotyping differentiated the isolates into 16 distinct ribotypes (similarity>93%), belonging to 17 different DuPont Identification Library Codes (DUP-IDs) clones. The Simpsons numerical index of discrimination was 0.911. Cluster analysis pointed out a high similarity among strains isolated from meat, fish, and vegetables of different origin. These results confirmed the existence of a widespread population of L. monocytogenes, characterized by highly related strains existing in different geographical areas. 65% of these strains belonged to lineage II (serotypes 1/2a and 1/2c), subtypes known to be associated with sporadic human listeriosis outbreaks. The remaining 35% of the isolates (serotypes 1/2b, 3b and 4b) were allocated to lineage I and belong to distinct clonal groups (DUP-ID 1038 and 1042), which again have been associated with several outbreaks of human listeriosis. Neither atypical profiles nor lineage III strains were found. EcoRI ribotyping was confirmed as a rapid and reliable method for L. monocytogenes typing, providing useful data for epidemiologic and clonality surveys of L. monocytogenes strains isolated from RTE foods.

Investigation of Salmonella enterica in Sardinian slaughter pigs: prevalence, serotype and genotype characterization

In order to improve the knowledge about the presence of Salmonella in pork meat in Sardinia (Italy), the prevalence and the sources of Salmonella at 5 pig slaughterhouses (slaughtered pigs and environment) were investigated and the isolates were characterised. A total of 462 samples were collected, 425 from pigs at slaughter and 41 from the slaughterhouse environment. Salmonella was isolated from 26/85 (30.5%) mesenteric lymph nodes, 14/85 (16.4%) colon contents, and from 12/85 (14.1%) carcasses and livers. Salmonella prevalence was 38% (8/21) in samples from surfaces not in contact with meat, and 35% (7/20) in those from surfaces in contact with meat. Thirty-one pigs were identified as carriers of Salmonella in lymph nodes and/or colon content, but of these, only 8 carcasses were positive. A total of 103 Salmonella isolates were serotyped and genotyped. Eight different serotypes were detected; the most common were S. Derby (44/103, 42.7%) and S. Typhimurium (24/103, 23.3%). The most prevalent S. Typhimurium phage type was DT193. Thirty-two isolates were found to be resistant to more than one antimicrobial (MDR). Pulse-field gel electrophoresis (PFGE) permitted the resolution of XbaI macrorestriction fragments of the Salmonella strains into 20 distinct pulsotypes. Combined application of a plasmid profiling assay (PPA) and PFGE gave useful additional information to assist in tracing the routes of Salmonella contamination in abattoirs. To reduce Salmonella prevalence some preventive measures should be encouraged: the origin of infected slaughter animals should be identified and direct and cross-contamination of carcasses should be avoided by adhering to HACCP principles in association with good hygiene procedures (GHP).

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.

Presence and molecular characterization of the major serovars of Listeria monocytogenes in ten Sardinian fermented sausage processing plants.

The aim of the present study was to investigate the occurrence of Listeria monocytogenes in ten Sardinian fermented sausage processing plants. A total of 230 samples were collected and 40 L. monocytogenes isolates were obtained and subjected to serotyping and investigated for the presence of ten virulence-associated genes using multiplex PCR assays. The isolates were further subjected to PFGE and investigated for their adhesion abilities in polystyrene microtiter plates. L. monocytogenes was found in 6% of food contact surfaces, in sausages at the end of acidification (3%) and ripening (8%). Serotyping revealed the presence of four serovars: 1/2c (37.5%), 1/2b (27.5%), 4b (22.5%) and 1/2a (12.5%). All virulence-associated genes were detected in 67.5% of the isolates. Isolates from processing environment, semi-processed and finished products showed high pulsotype diversity and the majority of isolates presented weak adhesion capability. The detection of the pathogen in fermented sausages confirms the ability of L. monocytogenes to overcome the hurdles of the manufacturing process.

Listeria monocytogenes in five Sardinian swine slaughterhouses: prevalence, serotype, and genotype characterization.

In a 3-year study (2008 to 2011) to estimate the prevalence and the contamination sources of Listeria monocytogenes in pork meat in Sardinia, Italy, 211 samples were collected from five Sardinian swine slaughterhouses: 171 samples from slaughtered pigs and 40 from the slaughterhouse environment. Fifty L. monocytogenes isolates were characterized by PCR-based serotyping, presence of virulence-associated genes, and pulsed-field gel electrophoresis restriction analysis. The overall prevalence of L. monocytogenes was 33% in swine carcasses, 7% in cecal material, 23% on meat contact surfaces, and 25% on noncontact surfaces. Only two serotypes were detected: 1/2c (78%) and 1/2a (22%). In all, based on the presence of virulence-associated genes, eight pathogenic profiles were detected. Only 42% of all isolates carried the full complement of virulence-associated genes and were allotted to profile 1. Six pulsed-field gel electrophoresis profiles persisted in the slaughterhouses; restriction profiles appeared to be specific to each plant.

Presence of Listeria monocytogenes in Mediterranean-Style Dry Fermented Sausages

The morphological, physiological and epidemiological features of L. monocytogenes, together with the severity of human listeriosis infections, make L. monocytogenes of particular concern for manufacturers of cold-stored “ready to eat” (RTE) foods. L. monocytogenes has been isolated from a wide variety of RTE foods and is responsible for several outbreaks associated with the consumption of RTE meat, poultry, dairy, fish and vegetable products. Although L. monocytogenes is among the most frequently-detected pathogens in dry fermented sausages, these products could be included in the category of RTE products in which the growth of L. monocytogenes is not favored and have rarely been implicated in listeriosis outbreaks. However, L. monocytogenes is highly difficult to control in fermented sausage processing environments due to its high tolerance to low pH and high salt concentration. In many Mediterranean-style dry fermented sausages, an empirical application of the hurdle technology often occurs and the frequent detection of L. monocytogenes in these products at the end of ripening highlights the need for food business operators to properly apply hurdle technology and to control the contamination routes of L. monocytogenes in the processing plants. In the following, through an up-to-date review of (personal and un-) published data, the main aspects of the presence of L. monocytogenes in Mediterranean-style dry fermented sausages will be discussed.

Mislabelling and species substitution in fishery products retailed in Sardinia (Italy), 2009-2014

Mislabelling and species substitution are major concerns for fishery products marketed in the EU. The present survey aimed to investigate the correct enforcement of the Community and National rules on the labelling and marketing of fishery products retailed in Sardinia (Italy) between 2009 and 2014. A total of 3000 labels for fresh unpacked fishery products have been considered. A total of 900 labels (30%) presented non-compliance concerning the wrong trade name, the wrong or missing information about the catch area and the production method. The highest percentage of mislabelling and species substitution has been detected in open-air markets (65%) and small-scale retail shops (40%) compared with the big supermarket chains (10%). The high percentage of non-compliances with the European and Italian legislation highlights the need to improve the essential information demanded by consumers on fishery products marketed in open-air markets and small-scale retail shops. While there are laws in place, it is unclear how effective they are and what type of penalties food business operators of open-air markets and small-scale retail shops may incur.

Listeria monocytogenes persistence in ready-to-eat sausages and in processing plants

Listeria monocytogenes is of major concern in the fermented meat products and is able to persist in their processing environments. The aim of the present work was to evaluate the virulence profile and the persistence capacity of L. monocytogenes strains isolated in Sardinian fermented sausages processing plants. Food (ground meat, sausages at the end of acidification and ripening stage) and environmental samples (a total of n. 385), collected from 4 meat processing plants located in Sardinia (Italy), were examined to detect L. monocytogenes presence. All the L. monocytogenes isolates were identified by polymerase chain reaction (PCR) method. A subset of strains was also characterised by multiplex PCR-based serogrouping, using the lmo0737, lmo1118, ORF2819 and ORF2110 genes. Three different multiplex PCRs were used to obtain the virulence profiles by the rrn, hlyA, actA, prfA, inlA, inlB, iap, plcA, plcB and mpl marker genes. Furthermore, in vitro biofilm forming ability and resistance to disinfectants were carried out on microtiter plate. The overall prevalence was 31.5% in food, and 68.5% in environmental samples. The prevalent serotype resulted 1/2c (43%), followed by 1/2a (40%), 4b (8.6%), and 1/2b (8.6%). The amplification products of the virulence genes were found in all the isolates with the following prevalence: 77.1% hlyA; 100% rrn; 100% prfA; 97.1% iap; 65.7% inlB; 88.6% inlA; 100% plcA; 100% plcB and 74.3% mpl. As for biofilm forming ability, 37.1% of the strains were positive and resulted weak producer, but all the isolates were sensible to disinfectants showing a reduction of L. monocytogenes growth after each incubation time. More appropriate technologies and application of measures of hygienic control should be implemented to prevent the L. monocytogenes growth and cross-contamination in salsiccia sarda processing plants.

Labelling and marketing of bivalve and gastropod molluscs retailed in Sardinia, Italy between 2009 and 2013

The aim of the present survey was to investigate the correct enforcement of the Community rules on the labelling and marketing of bivalve and gastropod molluscs retailed in Sardinia, Italy between 2009 and 2013. A total of 1500 packages and labels for live bivalve and gastropod molluscs were considered. A total of 375 labels (25%) presented non-compliance concerning the wrong trade name and additional wrong or missing information. The highest percentage of anomalous labels has been detected in small-scale retail shops (35%) and open-air markets (25%) compared with the big retailing chains (20%). The 5% of packages were not in compliance with the European Community rules on packaging of bivalve and gastropod molluscs. The high percentage of non-compliance with the European regulations on labelling results is a strong limitation for the consumers and highlights the need to improve the control system about labelling of seafood products.

A Rapid Colorimetric Method Reveals Fraudulent Substitutions in Sea Urchin Roe Marketed in Sardinia (Italy)

In recent years, besides the consumption of fresh sea urchin specimens, the demand of minimally-processed roe has grown considerably. This product has made frequent consumption in restaurants possible and frauds are becoming widespread with the partial replacement of sea urchin roe with surrogates that are similar in colour. One of the main factors that determines the quality of the roe is its colour and small differences in colour scale cannot be easily discerned by the consumers. In this study we have applied a rapid colorimetric method for reveal the fraudulent partial substitution of semi-solid sea urchin roe with liquid egg yolk. Objective assessment of whiteness (L*), redness (a*), yellowness (b*), hue (h*), and chroma (C*) was carried out with a digital spectrophotometer using the CIE L*a*b* colour measurement system. The colorimetric method highlighted statistically significant differences among sea urchin roe and liquid egg yolk that could be easily discerned quantitatively.