Enumeration of Escherichia coli and determination of Salmonella spp. and verotoxigenic Escherichia coli in shellfish (Mytilus galloprovincialis and Ruditapes decussatus) harvested in Sardinia, Italy.

The aim of the present study was to evaluate the occurrence of Salmonella spp., Verotoxigenic E. Coli (VTEC) and enumerate E. coli in shellfish (Mytilus galloprovincialis and Ruditapes decussatus) collected before and after depuration from two class B harvesting areas located in Sardinia (Italy). All the samples were analyzed for Salmonella spp. detection according to European Commission Regulation (EC) 2073/2005 and examined using the five tube Most-Probable-Number (MPN) method for enumeration of E. coli in accordance with the European Union reference method ISO 16649-3:2015. E. coli VTEC was investigated following a direct multiplex Polymerase Chain Reaction (PCR) screening test. The enumeration of E. coli met the European law limit for Class A areas of 230 MPN/100g. The averaged enumeration of E. coli in samples of M. galloprovincialis and R. decussatus collected at the harvesting time was 39 and 37 MPN/100 g respectively. The average contamination levels in samples collected after purification were 58 MPN/100g (M. galloprovincialis) and 32 MPN/100 g (R. decussatus). E. coli VTEC was not detected, on the contrary, Salmonella ser. Typhimurium was detected in one sample of M. galloprovincialis and in one sample of R. decussatus collected at the harvesting time. No significant associations were observed between E. coli levels in shellfish and environmental parameters of water or with the detection of Salmonella ser. Typhimurium in M. galloprovincialis and R. decussatus samples. Nevertheless, the occurrence of Salmonella ser. Typhimurium, involved in human infection outbreaks, should be considered a potential risk for consumers.

Occurrence, Seasonal Distribution, and Molecular Characterization of Vibrio vulnificus, Vibrio cholerae, and Vibrio parahaemolyticus in Shellfish (Mytilus galloprovincialis and Ruditapes decussatus) Collected in Sardinia (Italy).

In this study, we investigated the occurrence, seasonal distribution, and molecular characterization of pathogenic vibrios in Mediterranean mussels (Mytilus galloprovincialis) and grooved carpet shells (Ruditapes decussatus) from two harvesting areas of Sardinia (Italy). Samples collected before and after depuration were submitted for qualitative and quantitative determination of Vibrio spp. Vibrio spp. isolates were presumptively identified by means of biochemical methods. Identification and virulence profile of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus were performed by molecular methods. The prevalence of Vibrio spp. in M. galloprovincialis and R. decussatus was, respectively, 96 and 77%. The averaged enumeration (mean ± standard deviation) of Vibrio spp. in samples of M. galloprovincialis and R. decussatus collected at the harvesting time was 2.04 ± 0.45 and 2.51 ± 0.65 log CFU/g, respectively. The average contamination levels in samples collected after purification were 2.28 ± 0.58 log CFU/g (M. galloprovincialis) and 2.12 ± 0.67 log CFU/g (R. decussatus). Four potentially pathogenic V. parahaemolyticus isolates (tdh+ or trh+) were recovered from grooved carpet shells samples. No isolate was tdh+/trh+. The presence of potentially pathogenic vibrios in Sardinian waters strengthens the need for rational purification practices under controlled conditions to guarantee the protection of consumers.

Clonal relationship among Vibrio parahaemolyticus isolated from Mediterranean mussels (Mytilus galloprovincialis) and grooved carpet shells (Ruditapes decussatus) harvested in Sardinia (Italy).

The aim of the present study was to investigate the genetic variability of Vibrio parahaemolyticus strains isolated from naturally contaminated Mediterranean mussels (Mytilus galloprovincialis) and Grooved carpet shells (Ruditapes decussatus) from three harvesting areas of Sardinia (Italy) using a combination of different typing methods: traditional phenotypic systems and molecular techniques. Ninety-nine putative V. parahaemolyticus strains isolated from shellfish collected before and after purification were included in the study. Seventy-two isolates were confirmed as V. parahaemolyticus and were submitted to REP, ERIC and BOX PCRs. The combined dendrogram showed the similarity of the data set of the three typing methods and demonstrates how the different techniques grouped the strains in two clusters in accordance with each singular dendrogram. Several strains rendered a unique pattern regardless of the typing method, which indicates the high discriminatory power of the methods. Moreover, the use of multiple typing methods allowed a more accurate characterization of the genetic profiles of isolates and the identification of clones hardly revealed through the common techniques. The intraspecific typing of environmental V. parahaemolyticus can be of great interest in order to recognize clonal relationships between environmental contamination, foodborne disease, and geographical/temporal distribution of this pathogen. The comparative analysis focusing on the obtained genetic profiles supports the possibility for typing methods to discriminate strains with similar phenotypic profile, identifying the level of genetic correlation among the strains and the presence of genetic clones.

Prevalence, bioserotyping and antibiotic resistance of pathogenic Yersinia enterocolitica detected in pigs at slaughter in Sardinia.

The aims of the present study were to determine Yersinia enterocolitica prevalence in finishing pigs and piglets at slaughter and to characterize the isolates in terms of bioserotype, virulence profile, antimicrobial susceptibility and genetic diversity. During the years 2013-2014, nine pig slaughterhouses placed in Sardinia (Italy) were visited twice, in order to collect animal samples and scalding water. Overall, 609 samples respectively of tonsils (126), colon content (161), mesenteric lymph nodes (161) and carcass surfaces (161) were collected from 126 finishing pigs and 35 piglets. Moreover, 18 scalding water samples were collected. Samples were analyzed for the detection of Y. enterocolitica according to ISO 10273-2003 standard (with some modifications). With regard to finishing pigs, Y. enterocolitica was detected in 11.9% of colon content samples, 3.2% of tonsils and 2.4% of lymph nodes. In piglets, Y. enterocolitica prevalence was 8.6% in colon content and 2.8% lymph nodes samples. Y. enterocolitica was not detected from carcass surface samples of both finishing pigs and piglets and from scalding water samples. Isolates were bio- and serotyped, tested for the presence of four virulence genes by PCR (ail, ystA, ystB and inv) and for antimicrobial resistance by disc-diffusion method. Among 47 confirmed isolates, 33 (70.2%) belonged to bio-serotype 4:O3, 7 (14.9%) to bio-serotype 2/O:5 and 7 (14.9%) to bio-serotype 1A. Bio-serotype 1A was detected only in isolates of piglets' samples. In bio-serotype 4/O:3 isolates the most common virulence genes were ystA (97.0%), ail (84.8%) and inv (78.8%). In bio-serotype 2/O:5, ail, inv and ystA genes were detected in all of the isolates. All bio-serotype 1A isolates were ystB positive (lacking ail, inv and ystA). All isolates were susceptible to cefotaxime, ceftazidime, chloramphenicol, ciprofloxacin, enrofloxacin, gentamicin, nalidixic acid, sulphonamide, tetracycline and trimethoprim-sulphametoxazole. Resistances to ampicillin and cefalothin were the most common (100%), followed by amoxicillin/clavulanic acid (83.0%) and streptomycin (4.3%). Resistance to amoxicillin/clavulanic acid was detected in 57% of bio-serotype 4/O:3 isolates, 71% of bio-serotype 1A and 100% of bio-serotype 2/O:5 isolates. Two bio-serotype 4/O:3 isolates (6%) were resistant to streptomycin. Thirty-two pathogenic Y. enterocolitica isolates were tested by NotI-PFGE, which identified 5 patterns among bio-serotype 4/O:3 isolates and 2 patterns among bio-serotype 2/O:5 isolates. This study provides epidemiological data about human pathogenic Y. enterocolitica and highlight the role of pigs as a potential source of infection for the consumers in Sardinia.

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.

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.

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.

Salmonella Prevalence and Microbiological Contamination of Pig Carcasses and Slaughterhouse Environment.

In seven EC swine abattoirs Salmonella prevalence (ISO 6579/2002) and serotypes of 25 piglets, 61 finishing pigs (lymph nodes, colon content, carcass and liver surface) and slaughterhouse environments (scalding water, surfaces in contact with meat and not in contact with meat) were investigated. Moreover, aerobic colony count [total viable count (TVC); ISO 4833] and Enterobacteriaceae (ISO 21528-2) of piglets and finishing pigs' carcasses were evaluated, and the results compared with EU process hygiene criteria (Reg. EC 2073/2005). Salmonella was not isolated in any of the piglets samples. Prevalence differed between slaughterhouses (P<0.5), and Salmonella was isolated from 39 of 244 samples of finishing slaughtered pigs (15.9%) and from 4 of 45 environmental samples (8.9%). In pig samples, carcasses showed the highest prevalence (18%) followed by colon content (14.8%), lymph nodes (13%) and liver (1.6%). S. Anatum was the most prevalent serotype (71.8%), followed by S. Derby (33.3%), S. Bredeney (5%) and S. Holcomb (2.5%). Between environmental samples, S. Anatum (50%), S. Bredeney and S. Derby (25%) were identified. Total viable mean counts (log10 CFU/cm2) of carcass surfaces ranged from 4.6 and 5.7 for piglets, and from 4.6 and 5.9 for finishing pigs, while Enterobacteriaceae ranged between 1.1 and 5 for piglets and between 2.1 and 5.3 for finishing pigs. These results were not in compliance with EU performance criteria. Total aerobic viable counts and Enterobacteriaceae mean levels of environmental samples appeared critical, particularly referred to surfaces in contact with meat (splitting equipment) and indicated an inadequate application of good manufacturing and hygiene practices during slaughtering and sanitisation.

Detection and quantification of Vibrio parahaemolyticus in shellfish from Italian production areas.

Vibrio parahaemolyticus is a marine microorganism, recognized as an important cause of foodborne illness particularly in Asia, South America and United States. Outbreaks are rarely reported in Europe, but they can occur unexpectedly in relation, among other reasons, to the spread of highly virulent strains. It is known that the risk is proportional to exposure levels to pathogenic V. parahaemolyticus (i.e. carrying the tdh and/or the trh genes) but currently there is a lack of occurrence data for pathogenic V. parahaemolyticus in shellfish production areas of the Member States. In this study a total of 147 samples of bivalve molluscs, from harvesting areas of two Italian regions (Sardinia and Veneto) were analyzed for Escherichia coli and salmonella, according to Reg 2073/2005, and for detection and enumeration of total and toxigenic V. parahaemolyticus strains using a new DNA colony hybridization method. Environmental parameters (water temperature and salinity) were also recorded. Results of E. coli were consistently in agreement with the legislation limits for the harvesting class of origin and Salmonella was detected only in one sample. The average contamination levels for total V. parahaemolyticus were 84 and 73 CFU/g respectively for Sardinia and Veneto, with the highest value reaching 8.7 × 10(3)CFU/g. Nineteen samples (12.9%) resulted positive for the presence of potentially pathogenic V. parahaemolyticus strains, with levels ranging between 10 and 120 CFU/g and most of the positive samples (n=17) showing values equal or below 20 CFU/g. A significant correlation (r=0.41) was found between water temperature and V. parahaemolyticus levels, as well as with isolation frequency. The data provided in this study on contamination levels of total and potentially pathogenic V. parahaemolyticus, seasonal distribution and correlation with water temperature, will help in defining appropriate monitoring programs and post-harvest policies for this hazard, improving the management of the harvesting areas and the safety of bivalve molluscs.

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 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.

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.