Yersinia enterocolitica in slaughter pig tonsils: Enumeration and detection by enrichment versus direct plating culture

Tonsil samples from 139 slaughter pigs were examined for the presence of pathogenic Yersinia enterocolitica by enrichment procedures based on the standard method ISO 10273:2003. In addition, samples were tested by direct plating method to evaluate its efficiency compared to the enrichment culture methods and to quantify the level of contamination in porcine tonsils. In total, 52 samples (37.4%) were positive for pathogenic Y. enterocolitica, all belonging to bioserotype 4/O:3. Fifty out of the 52 positive samples (96.2%) were detected by direct plating. Enumeration showed an average concentration of 4.5 log₁₀ CFU g⁻¹ and 4.4 log₁₀ CFU g⁻¹ tonsil on Salmonella-Shigella-desoxycholate-calcium chloride (SSDC) and cefsulodin-irgasan-novobiocin (CIN) agar plates, respectively. The enrichment procedures recommended by the ISO 10273:2003 method were not optimal for the isolation of pathogenic Y. enterocolitica from pig tonsils: two days enrichment in irgasan-ticarcillin-potassium chlorate (ITC) broth resulted in an isolation rate of 84.6%, while 5 days enrichment in peptone-sorbitol-bile (PSB) broth recovered only 59.6% of positive samples. Reducing the enrichment time in PSB from 5 to 2 days resulted in a significantly higher recovery rate (94.2%) and might serve as an appropriate enrichment protocol for the isolation of pathogenic Y. enterocolitica from pig tonsils. Compared to enrichment culture methods, results based on direct plating can be obtained in a shorter time course and provide quantitative data that might be needed for further risk assessment studies.     

A real-time PCR for Detection of Pathogenic Yersinia enterocolitica in food combined with an Universal Internal Amplification Control System

A real-time PCR system with an internal amplification control was developed for detection of pathogenic Yersinia) enterocolitica in food samples. The chromosomally encoded ail gene was chosen as PCR target. Sequences of plasmid pUC19 served as target for the internal amplification control. The method was validated in combination with sample enrichment in PSB and TSB broth using different food matrices spiked with Y. enterocolitica and naturally contaminated slaughterhouse samples. The results of the real-time PCR with internal control were verified by the cultural method according to EN ISO 10273:2003. The sensitivity of the real-time PCR with internal control is about 5 genome copies per reaction. Artificial contamination of food samples resulted in a detection level of 5 cfu per 25 g Y. enterocolitica in food samples. 100% of porcine tonsils and about 22% meat from pig heads were contaminated. The screening of samples by PCR prior to cultural analysis allows focusing on positive samples in routine analysis. This could result in a higher detection rate by cultural analysis.     

A real-time PCR for Detection of Pathogenic Yersinia enterocolitica in food combined with an Universal Internal Amplification Control System

Summary:  A real-time PCR system with an internal amplification control was developed for detection of pathogenic Yersinia) enterocolitica in food samples. The chromosomally encoded ail gene was chosen as PCR target. Sequences of plasmid pUC19 served as target for the internal amplification control. The method was validated in combination with sample enrichment in PSB and TSB broth using different food matrices spiked with Y. enterocolitica and naturally contaminated slaughterhouse samples. The results of the real-time PCR with internal control were verified by the cultural method according to EN ISO 10273:2003. The sensitivity of the real-time PCR with internal control is about 5 genome copies per reaction. Artificial contamination of food samples resulted in a detection level of 5 cfu per 25 g Y. enterocolitica in food samples. 100% of porcine tonsils and about 22% meat from pig heads were contaminated. The screening of samples by PCR prior to cultural analysis allows focusing on positive samples in routine analysis. This could result in a higher detection rate by cultural analysis.

---

Zusammenfassung:  Für den Nachweis von pathogenen Yersinia enterocolitica wurde ein real-time PCR System mit interner Amplifikationskontrolle entwickelt. Das Nachweissystem für pathogene Y. enterocolitica basiert auf dem chromosomal kodierten ail-Gen. Als Zielsequenz der internen Amplifikationskontrolle dient eine Sequenz aus dem Plasmid pUC19. Zur Validierung der Methode wurden sowohl natürlich kontaminierte Proben aus einem Schlachthof als auch künstlich kontaminierte Proben verschiedener Lebensmittelmatrices verwendet. Die Anreicherung der Proben vor der molekularbiologischen Untersuchung erfolgte parallel in Tryptikase Soja-Bouillon (TSB) und in Pepton-Sorbit-Gallensalz-Bouillon (PSB). Die Ergebnisse der molekularbiologischen Untersuchungen wurden anschlie?end kulturell in Anlehnung an das Standardverfahren nach EN ISO 10273:2003 verifiziert. Die real-time PCR mit interner Amplifikationskontrolle weist eine Sensitivit?t von 5 Genomkopien pro Reaktionsansatz auf. Die Nachweisgrenze des Verfahrens, bestimmt anhand künstlich kontaminierter Proben, betr?gt etwa 5 KbE Y. enterocolitica pro 25 g Lebensmittel. Von den natürlich kontaminierten Proben aus einem Schlachthof waren die Tonsillen vom Schwein zu 100% mit pathogenen Y. enterocolitica kontaminiert, Schweinefleischabschnitte aus dem Kopfbereich wiesen einen Kontaminationsgrad von 22% auf. Ein Screening von Proben durch PCR erlaubt in der Routineanalytik die Fokussierung der kulturellen Analyse auf positive Proben. Dies k?nnte zu einer h?heren Nachweisrate durch das kulturelle Verfahren führen.

---

Received: March 5. 2008; accepted: March 14. 2008     

Genotypic and phenotypic virulence characteristics and antimicrobial resistance of Yersinia spp. isolated from meat and milk products

A total of 300 food samples including 180 milk and 120 meat products have been examined for the presence of Yersinia spp. using the ISO 10273 and the cold enrichment method. The overall prevalence of Yersinia spp. was 84 (28%). Yersinia enterocolitica was isolated from 18 (6%) of the 300 samples. The other Yersinia species were detected in the samples Yersinia rohdei 15 (5%), Yersinia intermedia 14 (4.7%), Yersinia pseudotuberculosis 12 (4%), Yersinia ruckeri 12 (4%), Yersinia mollaretii 5 (1.7%), Yersinia bercovieri 4 (1.3%), and atypical Yersinia spp. 4 (1.3%). The conventionally identified Y. enterocolitica strains were also confirmed by the 16S rRNA gene sequencing. All Y. enterocolitica strains biotyped as 1A had negative results in the phenotypic virulence tests. The 84 Yersinia strains were also examined genotypically for the presence of virulence genes. None of the Y. enterocolitica and other Yersinia strains contained the ail, ystA, yadA, and virF except only 1 Y. intermedia and 2 Y. enterocolitica strains that were found to be positive for ystB. Antimicrobial resistance of 84 Yersinia to 16 antimicrobial agents was determined by the disk diffusion method. All strains were sensitive to tobramycine and imipenem while resistant to clindamycin. Although 84.5% of the strains were resistant to at least 3 or more antimicrobial agents, 64.3% of them were resistant to 4 or more antimicrobial agents.     

Development and evaluation of DNA and RNA real-time assays for food analysis using the hilA gene of Salmonella enterica subspecies enterica

The objective of this study was the development of DNA and RNA real-time PCR methods for detection of food-borne Salmonella sp. as rapid alternatives to the traditional cultural method (ISO 6579, 2004) in fresh meat carcasses and processed meat samples. These PCR methods were based on the hilA sequence, with primers and hybridisation probes designed against this gene target. The primers and probes were evaluated for their efficiency and dynamic range and subsequently the specificity of the assay was tested using 106 Salmonella enterica subspecies enterica strains and 30 non-salmonellae strains. An internal amplification control (IAC) was also developed for incorporation. The optimum copy number of IAC was determined to be 500 copies per reaction. A complementary enrichment protocol was adapted from the existing standard ISO 6579:2004 and consisted of enrichment in Buffered Peptone Water (BPW) 22 ± 2 h and a second selective enrichment for 6 h in Rappaport Vassiliadis with Soya (RVS). The DNA and RNA-based real-time PCR protocols, were applied to meat samples inoculated with Salmonella enterica subspecies enterica strains, including swabs from meat carcasses and minced beef samples which were heat treated or frozen. The developed methods have the potential as useful alternatives to the standard ISO 6579:2004 method for the detection of Salmonella enterica subspecies enterica on carcass swabs and raw meat using hilA as a target. The DNA assay is a useful tool for the screening of meat samples in the abattoir within 3 days of slaughter or in a food production process and the RNA-based assay has the potential to detect viable Salmonella enterica subspecies enterica in ready-to-eat products.     

Incidence and pathogenicity of Yersinia spp. isolates from poultry in Spain

Forty eviscerated and refrigerated chicken carcasses were collected from retail outlets in León (Spain) and investigated for the presence of Yersinia spp. Following a two-step enrichment procedure (phosphate buffer saline was used as primary enrichment and bile oxalate sorbose as secondary enrichment), yersiniae were isolated on cefsulodin-irgasan-novobiocin (CIN) and MacConkey agar. Yersinia spp., Y. enterocolitica and Y. frederiksenii were detected in 26 (65%), 22 (55%) and six (15%) samples, respectively. The incidence ofYersinia and Y. enterocolitica was greater than that obtained in poultry meat by the majority of the consulted authors. Of the 68 Yersinia isolates, 52 were identified as Y. enterocolitica and 16 as Y. frederiksenii. Biotyping of Y. enterocolitica revealed that 45 (86·5%) strains corresponded to biotype 1A, and three (5·8%) to biotype 3. Four (7·7%) strains could not be classified. Two biotype 3 strains were found to be presumptively virulent according to the dye (Congo red and crystal violet) binding, low-calcium response, and pyrazinamidase activity techniques. It is suggested from results that the presence of Yersinia in poultry could represent a health risk for consumers in Spain. The education of people involved in production, processing and final preparation of poultry products are required in order to assure adequate cooking and to avoid cross-contamination.     

Detection of Yersinia spp. in meat products by enrichment culture, immunomagnetic separation and nested PCR

The prevalence of Yersinia enterocolitica in meat products was assessed by four methods: cold enrichment in trypticase soy broth (A), enrichment in modified Rappaport broth at 25 °C (B), concentration by immunomagnetic separation (C) and yadA nested PCR (D). Furthermore, the pathogenic potentials of the isolates were established by phenotypic and genotypic tests, and their genomic relationships were determined by pulsed-field gel electrophoresis (PFGE). A total of 238 samples were collected at retail level in the city of San Luis, Argentina, during the period 2007–2008. The highest Yersinia prevalence in meat products was observed by method D (92 positive samples), followed by methods A (13 positive samples) and C (5 positive samples); however, no isolation was obtained by method B. Fourteen Y. enterocolitica and 4 Yersinia intermedia strains were recovered by culture. All Y. enterocolitica 2/O:9 strains gave results related to virulence by phenotypic tests and exhibited the genotype virF⁺myfA⁺ail⁺ystA⁺. Two biotype 1A strains showed a genotype virF⁻myfA⁻ail⁺ystA⁺ystB⁺. The 14 Y. enterocolitica strains isolated during this work plus one reference strain were separated into 11 genomic types by PFGE. This genomic heterogeneity of the isolates shows the diversity of Y. enterocolitica strains in our region. It is the first time that IMS was used to search Y. enterocolitica strains from naturally contaminated meat products.     

Characterization of Yersinia enterocolitica and related species isolated from foods and porcine tonsils in the Netherlands

Yersinia enterocolitica and related species were isolated from a great variety of foods. High isolation rates were found in raw minced pork and beef, porcine tonsils and tongues, egg products, raw vegetables and surface water. Of 310 strains isolated 221 (71%) were identified as Y. enterocolitica and 89 as either Y. frederiksenii (12%), Y. intermedia (8%), Y. kristensenii (5%) or other Yersinia species (4%). The great majority (92%) of these strains were of biotype 1. Only 4% of the Yersinia isolates belonged to serotype 0 : 3 or 0 : 9, which are the main serotypes isolated from patients with yersiniosis in The Netherlands. Based on the results of an autoagglutination virulence assay and a test for calcium dependency at 37°C only the 0 : 3 and 0 : 9 strains isolated from porcine tonsils were potentially virulent. Most of the strains were isolated using cold enrichment in phosphate-buffered saline containing sorbitol and bile salts. However, the potentially virulent strains were all isolated using modified Rappaport broth.     

High prevalence of yadA-positive Yersinia enterocolitica in pig tongues and minced meat at the retail level in Finland

Polymerase chain reaction (PCR) was used to determine the prevalence of yadA-positive Yersinia enterocolitica in pig tongues and minced meat at the retail level in Finland and to confirm the yadA-positive Y. enterocolitica isolates recovered from the same samples using the conventional culture method. A total of 51 pig tongues purchased at 12 retail outlets and 255 minced meat samples purchased at 40 retail outlets in the Helsinki area were studied. The prevalence of Y. enterocolitica carrying the yadA gene was 92% in pig tongues and 25% in minced meat using PCR and 78% in tongues and 2% in minced meat with the culture method. The prevalence of yadA-positive tongues was higher (98%) when both PCR- and culture-positive results were included because Y. enterocolitica carrying the yadA gene could also be isolated in three PCR-negative tongue samples. In the minced meat samples, all PCR-negative samples were also culture-negative. With the culture method, 66 of 80 yadA-positive isolates in 38 tongues and all yadA-positive isolates (4) in four minced meat samples were recovered after selective enrichment. A total of 92 isolates of Y. enterocolitica bioserotype 4/O:3 in tongues and 5 isolates in minced meat were found, of which 13% in tongues and 20% in minced meat did not carry the yadA gene.     

Evaluation of ISO 10272:2006 standard versus alternative enrichment and plating combinations for enumeration and detection of Campylobacter in chicken meat

In the present study, we evaluate the recommended ISO 10272:2006 versus alternative procedures for Campylobacter enumeration and enrichment in naturally contaminated chicken meat samples (n = 49). Three enrichment media were evaluated; Bolton broth, Preston broth and CampyFood broth^® (bioMérieux SA, Marcy l’Etoile, France). In addition, three selective plating agars were compared; modified charcoal cefoperazone deoxycholate agar (mCCDA), CampyFood agar^® (CFA; bioMérieux SA) and Brilliance CampyCount agar^® (BCC; Oxoid, Basingstoke, England). Direct plating on CFA provided the highest number of Campylobacter positive samples (17/49); however this was not statistically different (P > 0.05) from numbers of positive samples recovered by direct plating on mCCDA (15/49) or BCC agars (14/49). Also, there was no significant difference between Campylobacter counts on the three compared media (P > 0.05). The coloured colonies of Campylobacter on CFA and BCC were easier to record and count than those on mCCDA. Enrichment of chicken meat samples in Bolton broth for 48 h and subsequent plating on CFA provided significantly higher (P < 0.05) Campylobacter detection compared to the other broth-agar combinations. Enrichment in Preston broth for 24 h followed by plating on mCCDA gave a higher number of positive samples (20/49) than 48 h enrichment in Bolton broth and plating on mCCDA (15/49). Enrichment in Bolton broth for 48 h followed by plating on CFA recovered 35% of samples below the limit for quantifications (<10 CFU/g, n = 34), as identified by direct plating on mCCDA. Compared to the current ISO method, some alternative combinations of enrichment and agar media could provide significantly better detection and enumeration of Campylobacter in chicken meat.     

Investigation of Yersinia enterocolitica, Yersinia enterocolitica-like bacteria,and thermotolerant campylobacters in Norwegian pork products

Altogether, Yersinia spp. were isolated from 41 (27.0%) of 152 samples of retail pork products. Thermotolerant campylobacters were not detected in this study. Yersinia spp. were recovered from 39 (30.7%) of the 127 raw pork samples and from two (8.0%) of the 25 cooked pork samples investigated. Sixty distinct strains were isolated. Y. enterocolitica comprised 58.3% of the total number, followed by Y. intermedia (20.0%), Y. kristensenii (18.3%), and Y. frederiksenii (3.3%). The serotyping scheme employed enabled us to type 91.7% of the strains. The most frequently isolated serotype was O:5 which comprised 20.0% of the total number, followed by O:7, 8 (11.7%), and O:11 (8.3%). Only one strain of O:3/biotype 4, the predominant human pathogen in Europe, was isolated. This was the only isolate which provoked positive reaction in the autoagglutination virulence assay. A second O:3 strain was classified as Y. kristensenii. Optimal recovery of Yersinia spp. was achieved using three-weeks cold enrichment in addition to a two-step procedure based on pre-enrichment in a low-selectivity medium (8 days, 4°C) followed by selective enrichment in modified Rappaport broth (4 days, 20–25°C). The results indicate that Yersinia spp. are more likely to be isolated from foods with a high level of coliforms than from foods with low coliform counts.     

A study of the growth of plasmid bearing and plasmid cured strains of antibiotic resistantYersinia enterocoliticaserotype 0:3 on refrigerated beef, pork and lamb

The growth of antibiotic-resistant strains of plasmid bearing (P+) and plasmid cured (P−) strains ofYersinia enterocoliticaserotype O:3 on minced and whole pieces of beef, pork and lamb at 0, 5 and 10°C was examined.Yersiniagrew on both meat forms and all species types at most storage temperatures examined. Similar growth rates were displayed by P+ and P− strains ofY. enterocoliticaunder each temperature and meat form, suggesting that the virulence plasmid had no significant effect on growth rates. It was noted, however, that initialYersiniagrowth on pork was slightly faster than on beef or lamb although the final numbers observed were slightly lower than on the other two meat types. The study demonstrated thatYersiniawas inhibited to a greater extent on pork than beef or lamb. This study has proposed that inhibition may be due to competition from the natural meat microflora, in particular, the Pseudomonads.     

Development and validation of a rapid real-time PCR based method for the specific detection of Salmonella on fresh meat

In this study, a combined enrichment/real-time PCR method for the rapid detection of Salmonella on fresh meat carcasses, was designed, developed and validated in-house following requirements outlined in ISO 16140:2003. The method included an 18 h non-selective enrichment in buffered peptone water (BPW) and a 6 h selective enrichment in Rappaport Vasilliadis Soya (RVS) broth, based on the traditional culture method, ISO 6579:2002. The real-time PCR assay included an internal amplification control (IAC), was 100% specific and was sensitive to one cell equivalent. The alternative method was validated against the traditional culture method and relative accuracy of 94.9%, sensitivity of 94.7% and specificity of 100% were determined using 150 fresh meat carcass swabs. This alternative method had a detection limit of 1–10 CFU/100 cm² for fresh meat carcass swabs and was performed in 26 h. Following further inter-laboratory studies, this alternative method could be suitable for implementation in testing laboratories for the analysis of carcass swabs.     

Prevalence of Yersinia enterocolitica in milk and dairy products and the effects of storage temperatures on survival and virulence gene expression

Yersinia enterocolitica was isolated from raw milk and dairy products from 10% of examined samples. The highest isolation rate was 22%, from raw milk, followed by 12%, 4% and 2% from fermented milk (Rayeb), pasteurised milk and ripened salted cheese, respectively. The virulence-associated genes ail and yst were detected in 30% and 10% of the isolates, respectively, while these genes were present simultaneously in 10% of the isolates. All the isolates showed susceptibility to gentamicin, ciprofloxacin and chloramphenicol, while only two of the isolates exhibited multidrug resistance. Storage of inoculated pasteurised milk at refrigeration (4 °C), freezing (−20 °C) and room (25 °C) temperatures revealed significant differences in Y. enterocolitica counts and relative expression of the two virulence genes. The isolation of potentially pathogenic Y. enterocolitica isolates from retail dairy products indicates risk to consumers; screening of prevalence, pathogenicity potential and antibiotic resistance is essential to implement control measures.     

Yersinia enterocolitica and Yersinia enterocolitica-like bacteria in Norwegian slaughter pigs

Yersinia spp. were isolated from the tonsils of 200 (43.4%) of 461 freshly slaughtered pigs. Y. enterocolitica comprised 92.7% of the total number of isolates, while Y. kristensenii, Y. intermedia, and Y. pseudotuberculosis comprised 6.3%, 0.5% and 0.5% respectively. Fifteen different serotypes were recognized. Serotype O:3 comprised 70.9% of the total number, followed by O:5 (6.8%), and O:6,30 (6.3%). Y. enterocolitica O:3/biotype 4, the predominating human pathogen in Europe, was isolated from 31.7% of the pigs examined. 67 (45.9%) of these isolates were potentially virulent as judged by the autoagglutination virulence assay. Three-week cold enrichment in a low-selectivity medium was the most efficient single method for recovery of Y. enterocolitica serotype O:3/biotype 4, Y. enterocolitica biotype 1, and Y. kristensenii. However, optimal recovery was achieved using a multi-method isolation technique, including a two-step procedure based on pre-enrichment followed by selective enrichment in a modified Rappaport broth.     

Pathogenic Yersinia enterocolitica 2/O:9 and Yersinia pseudotuberculosis 1/O:1 strains isolated from human and non-human sources in the Plateau State of Nigeria

Foodborne yersiniosis, caused by enteropathogenic Yersinia, especially Yersinia enterocolitica, is an important cause of diarrhea in developed countries, especially in temperate zones. Since studies concerning the presence of enteropathogenic Yersinia in humans and foods are rare in developing countries and tropical areas, human and non-human samples were studied in Plateau state of Nigeria to obtain information on the epidemiology of Y. enterocolitica and Yersinia pseudotuberculosis. Surprisingly, ail-positive Y. enterocolitica and inv-positive Y. pseudotuberculosis were isolated in Plateau state of Nigeria from several samples of human and non-human origin. Bioserotype 1/O:1 was the only Y. pseudotuberculosis type found. Y. enterocolitica belonging to bioserotype 2/O:9 was the dominating type found in most samples. Bioserotype 4/O:3 was isolated only from one pig and one sheep. Using PFGE, 5 genotypes were obtained among 45 Y. enterocolitica 2/O:9 strains with NotI, ApaI and XhoI enzymes and 3 among 20 Y. pseudotuberculosis 1/O:1 strains with NotI and SpeI enzymes. All human Y. pseudotuberculosis 1/O:1 strains were indistinguishable from pig, sheep or food strains. The dominating genotype of Y. enterocolitica 2/O:9 strains among humans was also found among strains isolated from pig, fermented cow milk and traditional intestine pepper soap samples.     

Pulsed field, PCR ribotyping and multiplex PCR analysis of Yersinia enterocolitica strains isolated from meat food in San Luis Argentina

The characterization of phenotypic and genotypic virulence markers of Yersinia enterocolitica strains belonging to biotypes (B) 1A, 2 and 3, mostly isolated from food in San Luis, Argentina, and the assessment of their genotypic diversity using PFGE and PCR ribotyping, were performed in our laboratory for the first time. Thirty five Y. enterocolitica strains, two reference strains and 33 strains isolated in our laboratory were studied. The presence of virF, ail, ystA, and myfA genes was investigated by multiplex PCR. The pathogenic potential of B1A strains, the most predominant biotype of Y. enterocolitica strains isolated from meat in our region, was investigated by simple PCR. Four B1A strains were positive for ystB gene. Four Y. enterocolitica 2/O:9 (bio/serotype) and two 3/O:5 strains isolated in our laboratory showed virulence-related results in the phenotypic tests and multiplex PCR. A good correlation between the expression of virulence markers and their corresponding genotypes was observed for most strains. Sixteen genomic types (GT) and 9 different intergenic spacer region (SR) groups were generated by PFGE and PCR ribotyping, respectively. In both cases the Y. enterocolitica 2/O:9 strains were separately clustered from 1A and 3/O:5 strains. Meat foods might be vehicles of transmission of pathogenic Y. enterocolitica strains in our region.     

Meta-analysis of the incidence of foodborne pathogens in Portuguese meats and their products

Meat and meat products are the main vehicles of foodborne diseases in humans caused by pathogens such as Salmonella spp., Campylobacter spp., Listeria monocytogenes, Yersinia enterocolitica, verotoxigenic Escherichia coli (VTEC) and Staphylococcus aureus. In order to prioritise research on those microbial hazards, a meta-analysis study was conducted to summarise available information on the presence of such pathogens in meats produced in Portugal. By using a logit-transformed proportion as effect size parameterisation, a number of multilevel random-effect meta-analysis models were fitted to estimate mean occurrence rates of pathogens, and to compare them among meat categories (i.e., bovine meat, broiler meat, pork, minced beef and minced pork), and among meat product categories (i.e., intended to be eaten cooked, to be eaten raw and cured meats). The mean occurrence rate of Campylobacter in Portuguese broiler meat (40%; 95% CI: 22.0–61.4%) was about ten times higher than that of Salmonella (4.0%; 95% CI: 1.4–10.8%); although these levels were comparable to current EU ranges. Nevertheless, in the other meat categories, the meta-analysed incidences of Salmonella were slightly to moderately higher than EU averages. A semi-quantitative risk ranking of pathogens in Portuguese-produced pork pointed Salmonella spp. as critical (with a mean occurrence of 12.6%; 95% CI: 8.0–19.3%), and Y. enterocolitica as high (6.8%; 95% CI: 2.2–19.3%). In the case of the Portuguese meat products, the non-compliance to EU microbiological criteria for L. monocytogenes (8.8%; 95% CI: 6.5–11.8%) and Salmonella spp. (9.7%; 95% CI: 7.0–13.4%) at sample units level, in the categories ‘intended to be eaten cooked’ and ‘to be eaten raw’, were considerably higher than EU levels for ready-to-eat products in comparable categories. S. aureus was the pathogen of greatest concern given its high occurrence (22.6%; 95% CI: 15.4–31.8%) in meat products. These results emphasised the necessity of Portuguese food safety agencies to take monitoring, and training actions for the maintenance of good hygiene practices during the production of the great variety of traditional meat products. This meta-analysis study also highlighted important gaps of knowledge, and may assist food safety authorities in the prioritisation of microbiological hazards, and the implementation of essential food safety assurance systems at primary production.     

Prevalence and characterization of pathogenic Yersinia enterocolitica in pig tonsils from different slaughterhouses

The prevalence of yadA-positive Yersinia enterocolitica was determined in 185 pig tonsils from nine slaughterhouses using both the PCR and culture method. The mean prevalence was 37%, varying from 13% to 45% when both PCR and culture-positive results were included. Of the 52 PCR-positive tonsil samples, 20 were culture-negative, while of the 48 culture-positive, 16 were PCR-negative. Using the culture method, Y. enterocolitica belonging to the bioserotype 4/O:3 was found in 61 tonsils, of which 48 were yadA-positive. Type 4/O:3 was the only pathogenic bioserotype found in this study. Most of the yadA-positive samples (85%) were recovered already after overnight enrichment. A total of 61 isolates, including 13 yadA-negative isolates from different samples, were characterized with PFGE. UsingNotI and XbaI, 20 and 17 PFGE patterns were obtained, respectively. Although the patterns were not identical, most of them played only minor deviations. A total of 26 pulsotypes, defined by combination of the various NotI andXbaI digestion profiles, were observed. Two to eight different pulsotypes were observed in each slaughterhouse, The most common pulsotypes, 1a and 4g, were found in 36% and 20% of the tonsils, respectively and these pulsotypes were widely distributed to most of the slaughterhouses. The pulsotype 1a was identified in eight out of nine slaughterhouses and the pulsotype 4g in seven slaughterhouses.     

Detection and prevalence of pathogenic Yersinia enterocolitica in refrigerated and frozen dairy products by duplex PCR and dot hybridization targeting the virF and ail genes

Pathogenic Yersinia enterocolitica is involved in yersiniosis through expression of chromosome-borne or plasmid-borne virulence factors. Yersinia enterocolitica is a cold-tolerant pathogen frequently isolated from refrigerated or frozen foods. However, little attention has been focused on the prevalence of pathogenic Y. enterocolitica in refrigerated or frozen dairy samples in China. In this study, we developed a new duplex PCR targeting the plasmid-borne virF gene and chromosome-borne ail gene for detection of pathogenic Y. enterocolitica isolates. We established a detection limit for the duplex PCR of 6.5 × 10² cfu/mL in artificially contaminated dairy samples. In addition, the duplex PCR could detect directly 4.5 to 5.7 cfu of Y. enterocolitica in 5 mL of brain heart infusion broth after 6 h of enrichment at 28°C. A newly developed dot hybridization assay further confirmed specificity of the duplex PCR for detection of virulent Y. enterocolitica. Furthermore, 13 Y. enterocolitica and 5 pathogenic strains, from 88 commercial frozen or refrigerated dairy products, were detected successfully by the China National Standard method (GB/T4789.8-2008) and the duplex PCR, respectively. Finally, biotypes and serotypes of pathogenic Y. enterocolitica strains were further characterized. The duplex PCR developed here is reliable for large-scale screening, routine monitoring, and risk assessment of pathogenic Y. enterocolitica in refrigerated or frozen dairy products.