Francesca Piras

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.

Influence of Temperature, Source, and Serotype on Biofilm Formation of Salmonella enterica Isolates from Pig Slaughterhouses.

Quantitative assessment of in vitro biofilm formation by 40 Salmonella enterica isolates isolated in pig abattoirs from animal and environmental sources (surfaces in contact and not in contact with meat) and classified in eight seroytpes was carried out by using a microtiter plate assay with spectrophotometric reading (optical density at 620 nm). Biofilm-forming ability was statistically correlated with the temperature of incubation (22 and 35°C), the source of the isolates, and the antimicrobial resistance profile. After incubation at 35°C, 9 isolates (22.5%) were classified as weak biofilm producers. After incubation at 22°C, 25 isolates (62.5%) were classified as weak producers and 3 (7.5%) as moderate producers. The quantity of biofilm formed after incubation at 22°C was significantly higher (P < 0.01) than at 35°C. This result is notable because 22°C is a common temperature in meat processing facilities and in slaughterhouses. At 35°C, isolates detected from surfaces in contact with meat showed significantly higher (P < 0.1) optical density values compared to isolates from other samples, highlighting the risk of cross-contamination for carcasses and offal. No correlation was detected between quantity of biofilm and serotype or between biofilm formation and resistance to antimicrobials.

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.

Occurrence, Characterization, and Antimicrobial Susceptibility of Salmonella enterica in Slaughtered Pigs in Sardinia.

The aim of this study was to determine Salmonella occurrence in slaughtered finishing pigs and piglets and in slaughterhouse environment in order to characterize the isolates with phenotypical (antimicrobial testing) and molecular (PFGE, MLVA) methods. Nine slaughterhouses located in Sardinia were visited. Six hundred and eight samples collected from 106 pigs and 108 environmental samples were collected and analyzed. Salmonella was isolated in 65 of 504 (12.9%) samples from finishing pigs, with an occurrence of 15.1% in colon content, 12.7% in lymph nodes and liver, and 11.1% in carcass surface samples. Salmonella was never detected in piglets. The combined results of serotyping and PFGE showed a possible self-contamination in 71.5% of Salmonella positive carcasses of lymph nodes and/or colon content carriers, pointing out the role of healthy pigs for carcass contamination. A significantly higher (P < 0.05) occurrence was detected in finishing pigs of EC countries origin (23%) than in pigs of local farms (8%). Salmonella was also detected in 3.7% of environmental samples. The most prevalent serovar was S. Anatum, followed by S. Rissen, S. Derby, and monophasic S. Typhimurium. Resistance to at least 3 antimicrobial was observed in 97.1% of strains and 7 different patterns of multiple resistance were identified. The most common resistance was detected against sulphonamide compounds. A strict slaughterhouse application of hygiene standards is essential to control the risk of Salmonella contamination.

Production of farmstead lactose-free Pecorino di Osilo and ricotta cheeses from sheep’s milk

The present work was aimed to define and validate farmstead production of lactose-free Pecorino di Osilo cheese, fresh ricotta cheese, and salted and smoked ricotta cheese (Ricotta mustia). The enzymatic activity of the commercial preparation containing lactase (1.1 g/mL), preliminarily tested using a spectrophotometric titration, showed activity equal to 4950±40 neutral lactase unit/g. The amount of lactase required to obtain the lactose-free milk was then established in triplicate laboratory trials, by adding the enzyme at concentrations of 0.7, 0.9 and 1.1 g/L in flasks containing 160 mL of raw sheep’s milk. Samples were incubated under conditions expected during milk storage and cheese-making. The residual lactose content in milk was determined by enzymatic method. The addition of lactase at concentration of 1.1 g/L of milk reduced the lactose concentration below the limit of detection (LOD) of 0.06 g/L. The procedure was validated at a dairy farm, using three different batches of bulk raw sheep’s lactose-free milk that were transformed into Pecorino di Osilo cheese. The resulting whey was used to produce fresh ricotta and Ricotta mustia cheese. Raw milk and whey samples were always below lactose detection limit. The residual lactose was measured in Pecorino di Osilo cheese, after 24 hours and 30 days from production; in fresh ricotta cheese, after 48 hours; in Ricotta mustia cheese after 7 days. The determination of lactose content in cheese samples was conducted by a gas chromatography-flame ionization detection method, which showed a LOD and limit of quantification respectively of 1.8 and 5.6 mg/kg for cheese, and 1.35 and 4.2 mg/kg for both ricotta cheeses. The lactose concentration was always below the relevant LOD values in all samples. The mean concentration of galactose and glucose were respectively 13,000±2000 and 11,000±2000 mg/kg in fresh Pecorino di Osilo, 1100±300 and 1200±300 mg/kg in fresh ricotta, and 950±400 and 750±250 mg/kg in Ricotta mustia. The results of the present study showed that the production of farmstead lactose-free Pecorino di Osilo cheese and ricotta cheeses from raw sheep’s milk is easily achievable. The main issue for farmstead production of artisanal lactose-free products is the implementation of permanent procedures based on hazard-analysis and critical control principles aimed at guaranteeing the effectiveness of the process and at acquiring analytical evidences to demonstrate the fulfilment of law requirements for labelling.

Occurrence of nematodes of the genus Anisakis in Mediterranean and Atlantic fish marketed in Sardinia

Anisakiasis is a gastrointestinal fish-borne zoonosis caused by the ingestion of third stage larvae of the genus Anisakis. Between January and December 2013, 1112 specimens of four commercial fish species (Engraulis encrasicolus, Merluccius merluccius, Scomber colias and Trachurus mediterraneus) marketed in Sardinia (Italy) were examined for Anisakis sp. The overall prevalence of Anisakis spp larvae was 39.9%, all morphologically identified as Type I. Scomber colias showed the highest prevalence (100%), followed by M. merluccius (Atlantic 91.0%, Mediterranean 71.2%), T. mediterraneus (32.7%) and E. encrasicolus (25.9%). All the larvae found in Mediterranean hosts were genetically identified as Anisakis pegreffii, whereas 90.0% of the larvae found in the Atlantic M. merluccius belonged to Anisakis simplex sensu stricto and 10.0% to A. pegreffii. The mean abundance of Anisakis sp. larvae was positively correlated with fish size in E. encrasicolus, Atlantic M. merluccius and local M. merluccius. The prevalence of infection was greater in the body cavity (37.9%) than in the edible muscle (9.4%). However, 1.8% of the examined fish were infected exclusively in the muscle. Therefore, the risk associated to the consumption of raw or undercooked fishery products poses the need of measures such as visual inspection and preventive treatments to guarantee consumers’ health.

Detection of pathogenic Yersinia enterocolitica in slaughtered pigs by cultural methods and real-time polymerase chain reaction

Healthy pigs carrying pathogenic to human Yersinia enterocolitica strains are the main source of entry into slaughterhouse, where cross-contamination of carcasses can happen. The aim of this work was to determine Y. enterocolitica prevalence in slaughtered pigs, investigating the presence of carriers in relation to carcass contamination. A total of 132 pig samples (tonsils, mesenteric lymph nodes, colon content, carcass surface) were collected from 4 Sardinian slaughterhouses. All the samples were examined by the ISO 10273:2003 method, and the prevalence was also determined by direct plating on CIN Agar. Moreover, to detect the ail positive Y. enterocolitica strains in enrichment broths and isolates a real-time polymerase chain reaction (PCR) was applied. Y. enterocolitica prevalence was 19% with direct plating and 12% with enrichment methods. Carcass surfaces and tonsils prevalence was 5.30% by direct plating, and 5.3% and 2.2%, respectively, by enrichment method. Tonsil samples showed an average contamination level of 3.2×103 CFU/g, while the mean value on carcass was 8.7×102 CFU/g. An overall prevalence of 9.8% of ail positive Y. enterocolitica broths was detected by RT-PCR, that found a higher prevalence in tonsils (7.5%) with respect to cultural methods, confirming the greater sensitivity of this technique when applied for tonsils and faeces samples. The results show a relatively low pathogenic Y. enterocolitica prevalence in pigs slaughtered in Sardinia. Good hygiene measures should be applied at slaughterhouse in order to prevent the entry of carriers and control carcass contamination.