Anna Mureddu

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).

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.

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.

Prevalence of Verocytotoxin-Producing E. coli in Sheep Meat at a Slaughterhouse

Sheep play important roles in the spread of pathogenic Escherichia coli that can cause severe diseases in humans. The aim of the present study was to evaluate the prevalence of verocytotoxin-producing E. coli (VTEC) in various samples from Sarda sheep using a molecular screening test and to define the virulence profiles of isolates using multiplex PCR. A total of 380 different ovine samples (fleece, carcass surface, and mucosal gut) were analyzed by direct PCR screening for the presence of stx 1 e stx 2 genes. Virulence factors (stx 1, stx 2, and eae) from the strains, isolated by immunomagnetic separation (IMS)-based cultivation techniques (e.g., CT-SMAC, CT-RMAC, and EHLY agar), were determined by multiplex PCR. An overall prevalence of 11.1% (adults 14%, lambs 7.8%) was found by direct PCR. VTEC occurrence was 18.9% in fleece, 14.7% on carcasses, and 10.5% in mucosal gut. According to the multiplex-PCR results, the following values were obtained: 43.4% VTEC (stx 1 , stx 2 , stx 1 + stx 2 , stx 2 + eae, and stx 1 + stx 2 + eae), 30.3% EPEC (stx 1 –/stx 2 –/eae+), and 26.4% negative for all genes examined (stx 1 –, stx 2 –, eae–).

Evaluation of short purification cycles in naturally contaminated Mediterranean mussels ( Mytilus galloprovincialis ) harvested in Sardinia (Italy)

The aim of the present study was to investigate the effect of short purification cycles on the safety of naturally contaminated Mytilus galloprovincialis from harvesting areas of the Gulf of Olbia (Sardinia, Italy). Samples from ten batches of mussels were collected before, during and after purification treatment at two purification centres (A-B). All the samples were analysed for Escherichia coli and Salmonella spp according to Council Regulation (EC) 2285/2015. Detection and enumeration of Vibrio spp were performed according to previously published methods. Presumptive identification of Vibrio spp isolates were performed by means of conventional biochemical tests and polymerase chain reaction. The presence of Hepatitis A virus was detected by nested reverse transcriptase-polymerase chain reaction. Environmental parameters (water temperature and salinity) were also recorded. The results of Escherichia coli counts showed the overall efficacy of the short purification cycles; a purification cycle of 8 h led to a rapid decline in the concentration. The decrease in Escherichia coli counts does not correlate with the presence of naturally occurring vibrios, the decline of which occurs at an even slower rate. The average contamination levels for Vibrio spp before purification were 8.20 ± 0.47 and 7.99 ± 0.62 Log10 CFU/g in samples collected at purification plants A and B, respectively. After purification, the average contamination levels were 8.10 ± 0.60 Log10 CFU/g at purification plant A and 7.85 ± 0.57 Log10 CFU/g at purification plant B. The contaminated samples revealed the presence of Vibrio alginolyticus (n=21), Vibrio fluvialis (n=12), Vibrio cholerae (n=4), Vibrio parahaemolyticus (n=2) and Vibrio vulnificus (n=1). The Vibrio parahaemolyticus isolates carried the tdh or the trh genes. None of the isolates was tdh+/trh+. Salmonella spp and Hepatitis A virus were not detected. The adoption of short purification cycles for Mytilus galloprovincialis in the presence of pathogenic vibrios might not be sufficient to guarantee the safety of consumers.

Determination of Salmonella spp., E. coli VTEC, Vibrio spp., and Norovirus GI-GII in Bivalve Molluscs Collected from Growing Natural Beds in Sardinia (Italy)

The aim of the present study was to evaluate the presence of Salmonella spp., verotoxigenic E. coli (VTEC), Vibrio spp., and Norovirus GI-GII in bivalve molluscs, cockles, and European grooved carpet shells (Cerastoderma spp. and Ruditapes decussatus) collected from a class B growing natural bed in Sardinia (Italy). All of the samples were analysed for Salmonella spp. detection according to European Commission Regulation (EC) 2285/2015. Detection and enumeration of Vibrio spp. were performed according to previously published methods. Presumptive identification of Vibrio spp. isolates was performed by means of conventional biochemical tests. E. coli VTEC was isolated following a direct multiplex polymerase chain reaction (PCR) screening test. Norovirus GI and GII were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). No Salmonella spp. were detected. The prevalence of Vibrio spp. was 90%, and the average contamination levels were 3.19 ± 1.07 and 2.84 ± 0.31 Log10 cfu/g in cockles and European grooved carpet shells, respectively. The prevalence of E. coli VTEC was 6.6%. All of the isolates showed a complete pathogenicity profile. The presence of Norovirus was highlighted in 25% of European grooved carpet shells samples. Results showed the typical microbiological profile of bivalve molluscs collected from backwaters and confirmed the capability of shellfish to accumulate E. coli VTEC, pathogenic vibrios, and Norovirus. The presence of such pathogens in shellfish is of major concern for the safety of consumers.

SHORT FOOD SUPPLY CHAIN IN SARDINIA: FORMS OF COOPERATION BETWEEN PRIMARY PRODUCERS AND FINAL CONSUMERS

The diffusion of the short food supply chains in Sardinia, at present, is not the same as in other Italian regions. Several structural and economic conditions of the Regional food supply chain, prevent an extensive diffusion. Forms of cooperation between primary producers and final consumers, as the responsible buying groups, will promote the shortening of the food supply chain, offering to the primary producers an alternative way of marketing for the wide array of locally grown farm produce.