Evaluation of the ISO 10273:2003 method for the isolation of human pathogenic Yersinia enterocolitica from pig carcasses and minced meat

Pig carcass swabs (n = 254) and minced meat samples (n = 82) were examined for pathogenic Yersinia enterocolitica using different routinely used enrichment protocols. All samples were obtained in the context of the official Yersinia monitoring program in Belgium. In total, 28 carcasses (11.0%) were contaminated with Y. enterocolitica bioserotype 4/O:3 and one (0.4%) with bioserotype 2/O:9. Four minced meat samples (4.9%) tested positive for Y. enterocolitica bioserotype 4/O:3. Using the ISO 10273:2003 method, eight out of the 29 Yersinia-positive carcasses (27.6%) and none of the contaminated minced meat samples (0.0%) were detected. Reducing the enrichment time in PSB from 5 to 2 days increased the number of positive samples. Overall, enrichment in PSB at 25 °C recovered more positive carcasses and minced meat samples than selective enrichment and cold enrichment. As the exclusive use of the ISO 10273:2003 method results in a strong underestimation of Y. enterocolitica positive carcasses and minced meats, efforts are needed to optimize the current version of the ISO method. In addition, isolation of pathogenic Y. enterocolitica requires experience and the use of a stereomicroscope to avoid false negative results.     

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.

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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.

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

High bacterial contamination of pig tonsils at slaughter

Food-borne zoonoses have a major health impact in industrial countries. Campylobacter spp., Salmonella enterica, Yersinia enterocolitica and Listeria monocytogenes are high-risk food-borne zoonotic hazards in finishing pigs. The objectives of this work were (1) to study the isolation rate of pathogenic Y. enterocolitica, Salmonella spp., Campylobacter spp. and L. monocytogenes in the tonsils and feces and (2) to determine the number of mesophilic aerobic bacteria (MAB) and Escherichia coli in the tonsils of fattening pigs at slaughter. The samples, which were collected from one slaughterhouse on five occasions, originated from 50 pigs and 15 farms. The number of MAB varied from 6.40 to 7.82 log₁₀ CFU/g and E. coli from 4.38 to 6.53 log₁₀ CFU/g. Additionally, 31 (62%) of the tonsils were colonized with Y. enterocolitica and 16 (32%) with L. monocytogenes. Campylobacter spp. were more frequently excreted in feces and only 3 (6%) of the pigs carried Campylobacter spp. in the tonsils. No Salmonella spp. were isolated. The pig tonsils were shown to be colonized with a high number of bacteria including E. coli, which is the most important indicator for fecal contamination, and with Y. enterocolitica and L. monocytogenes, which are important food-borne pathogens. This study demonstrates that the tonsils are highly contaminated with micro-organisms and can be a very important source of contamination in the slaughterhouse.     

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.     

Comparison of methods for the isolation of Yersinia enterocolitica from porcine tonsils and pork

Three enrichment procedures and three plating media were evaluated for their efficiency in isolating Yersinia enterocolitica from porcine tonsils and pork. Cold enrichment in phosphate-sorbitol-bile medium (PBSSB) with alkali treatment before plating resulted in higher isolation rates than enrichment in modified Rappaport broth (MRB) and bile-oxalate-sorbose broth (BOS). Post-enrichment alkali treatment gave a considerable increase in isolation rate with all the enrichment media tested. A sample inoculum of 10 g showed a better recovery than 0.2 g. Cefsulodin-irgasan-novobiocin (CIN) agar was slightly better than MacConkey agar and MacConkey agar + Tween 80 as isolation medium for Yersinia spp. from porcine tonsils. Most of the serotype 0:3 and 0:9 strains were isolated after enrichment in MRB and PBSSB. Enrichment in PBSSB resulted in the isolation of some other potentially virulent Yersinia strains. For the isolation of Yersinia spp. from pork and porcine tonsils an inoculum of 10 g, parallel use of MRB and PBSSB, post-enrichment alkali treatment and isolation on CIN agar is recommended.     

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.     

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.     

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.     

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.     

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.     

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.     

Occurrence of Bacillus cereus and Yersinia enterocolitica in South African retail meats

Ground beef, chicken broilers and processed meats (Vienna sausages, ham, salami) obtained from supermarkets in the Pretoria area (South Africa) were tested for the presence of Bacillus cereus and Yersinia enterocolitica. B. cereus counts were carried out on Bacillus cereus -selective agar and confirmed by the rapid confirmation procedure. Y. enerocolitica was isolated on Y. enterocolitica -selective agar. Total aerobic counts were performed on all the samples. Out of 51 samples, B. cereus was recovered from one broiler, three salami and five Vienna sausage samples, but not from gound beef or ham samples. The levels of detection ranged from log₁₀1·0 to log₁₀3·1 g⁻¹sample. Y. enterocolitica occurred in eight ground beef, eight broiler, one Vienna sausage and two ham samples. Our results emphasize the potential hazard of the presence of B. cereus and Y. enterolitica in meat and poultry products in South Africa. This necessitates the implementation of methods to prevent the entry or proliferation of these pathogens in meat products.     

Microbiological quality of fresh lettuce from organic and conventional production

Previously there was no available information on the levels of indicator bacteria and the prevalence of pathogens in fresh lettuce grown in organic and conventional farms in Spain. A total of 72 lettuce samples (18 farms for 4 repetitions each) for each type of the agriculture were examined in order to assess the bacteriological quality of the lettuces, in particular the prevalence of selected pathogens. The lettuce samples were analyzed for the presence of aerobic mesophilic, psychrotrophic microorganisms, yeasts and moulds, Enterobacteriaceae, mesophilic lactic acid bacteria, Pseudomonas spp. and presumptive Escherichia coli, Salmonella spp. and Listeria monocytogenes. The mean aerobic mesophilic counts (AM) were 6.35 ± 0.69 log₁₀ cfu g⁻¹ and 5.67 ± 0.80 log₁₀ cfu g⁻¹ from organic and conventional lettuce, respectively. The mean counts of psychrotrophic microorganisms were 5.82 ± 1.01 log₁₀ cfu g⁻¹ and 5.41 ± 0.92 log₁₀ cfu g⁻¹ from organic and conventional lettuce, respectively. Yeasts and moulds (YM) mean counts were 4.74 ± 0.83 log₁₀ cfu g⁻¹ and 4.21 ± 0.96 log₁₀ cfu g⁻¹ from organic and conventional lettuce, respectively. Lactic acid bacteria (LAB) were present in low numbers and the mean counts were 2.41 ± 1.10 log₁₀ cfu g⁻¹ and 1.99 ± 0.91 log₁₀ cfu g⁻¹ from organic and conventional lettuce, respectively. Pseudomonas spp. mean counts were 5.49 ± 1.37 log₁₀ cfu g⁻¹ and 4.98 ± 1.26 log₁₀ cfu g⁻¹ in organic and conventional lettuce, respectively. The mean counts for Enterobacteriaceae were 5.16 ± 1.01 log₁₀ cfu g⁻¹ and 3.80 ± 1.53 log₁₀ cfu g⁻¹ in organic and conventional lettuce, respectively. E. coli was detected in 22.2% (16 samples) of organic lettuce and in 12.5% (9 samples) of conventional lettuce. None of the lettuce samples was positive for E. coli O157:H7, L. monocytogenes and Salmonella spp. From the samples analyzed by principal component analysis (PCA) a pattern with two different groups (conventional and organic) can be observed, being the highest difference between both kinds of samples the Enterobacteriaceae count.     

The influence of headspace and dissolved oxygen level on growth and haemolytic BL enterotoxin production of a psychrotolerant Bacillus weihenstephanensis isolate on potato based ready-to-eat food products

The major objective of this study was to determine the influence of the initial headspace and dissolved O₂ level and vacuum packaging on growth and diarrhoeal enterotoxin production by Bacillus weihenstephanensis on potato based ready-to-eat food products. In general, the lower the initial headspace or dissolved O₂ level the slower the maximum growth rate (μ_max, log₁₀ CFU g⁻¹ d⁻¹), the longer the lag phase duration (λ, d) and the smaller the maximum population density (N_max, log₁₀ CFU g⁻¹) became. The slowest μ_max, the longest λ and the smallest N_max were generally found for growth under vacuum packaging. This implies shorter shelf-lives will occur at higher initial headspace or dissolved O₂ levels as the growth of B. weihenstephanensis to the infective dose of 10⁵ CFU g⁻¹ in such atmospheres takes a shorter time. Significant consumption of dissolved O₂ only occurred when growth shifted from the lag to the exponential phase and growth generally transitioned from the exponential to the stationary phase when the dissolved O₂ levels fell below ca. 75 ppb. Diarrhoeal enterotoxin production (determined via detection of the L2 component of haemolytic BL) was similar for growth under initial headspace O₂ levels of 1-20.9%, and was only reduced when growth took place under vacuum packaging. The reduction in L2 production when growth took place under vacuum was most probably related to the low final cell densities observed under this condition. Both growth and L2 production were inhibited over a 32-day incubation period at 7 °C by 40% CO₂ irrespective of the headspace or dissolved O₂ levels. The results illustrate the importance of residual O₂ and CO₂ on the shelf-stability and safety of modified atmosphere packaged potato based ready-to-eat food products with regards to B. weihenstephanensis.     

Thermal inactivation of Yersinia enterocolitica in pork slaughter plant scald tank water

The objective of this study was to establish the time–temperature combinations required to ensure the thermal inactivation of Yersinia enterocolitica during scalding of pork carcasses. A 2 strain cocktail of Y. enterocolitica (bioserotypes 2/O:5,27 and 1A/O:6,30) was heat treated at 50, 55 and 60 °C in samples of scald tank water obtained from a commercial pork slaughter plant. Samples were removed at regular intervals and surviving cells enumerated using (i) Cefsulodin–Irgasan–Novobiocin Agar (CIN) supplemented with ampicillin and arabinose and (ii) Tryptone Soya Agar (TSA), overlaid with CIN agar with ampicillin and arabinose. The data generated was used to estimate D- and z-values and the formula D_x = log^− 1(log D₆₀ − ((t₂ − t₁)/z)) was applied to calculate thermal death time–temperature combinations from 55 to 65 °C. D₅₀, D₅₅ and D₆₀-values of 45.9, 10.6 and 2.7 min were calculated from the cell counts obtained on CIN agar, respectively. The corresponding D-values calculated from the TSA/CIN counts were 45.1, 11 and 2.5 min, respectively. The z-value was 7.8. It was concluded that a time–temperature combination of 2.7 min at 60 °C is required to achieve a 1 log reduction in Y. enterocolitica in pork scald tank water. The predicted equivalent at 65 °C was 0.6 min. This study provides data and a model to enable pork processors to identify and apply parameters to limit the risk of carcass cross-contamination with Y. enterocolitica in pork carcass scald tanks.     

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.     

Detection of Pathogenic Yersinia enterocolitica in Meat using Real-Time PCR

Yersinia enterocolitica is an important zoonosis, which can cause disease in humans and animals. The culture methods available for detection of Y. enterocolitica in food samples are time-consuming and seldom successful. Using DNA-based methods, like PCR, this pathogen can be detected more rapidly and with greater sensitivity. The aim of this study was to establish a rapid and accurate real-time PCR method to detect pathogenic Y. enterocolitica in pork. The chromosomal ail−gene, which is only present in pathogenic Y. enterocolitica strains, was used as DNA target for the 5' nuclease PCR assay. The probe was labelled at the 5' end with the fluorescent reporter dye (FAM) and at the 3' end with the quencher dye (TAMRA). A 2-step protocol with 45 cycles was used in a multicolour real-time PCR detection system. A Ct value over 40 indicated a negative result. The DNA extraction procedure for the natural samples was rapid and simply. This qualitative real-time PCR method was shown to be specific and sensitive. Detection rate of ail-positive Y. enterocolitica in 200 pig tonsils was 88 % and 35 % with PCR and culture methods, respectively. When 100 raw pork samples were studied, 7 were positive with PCR and all were culture negative.     

Evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Vibrio parahaemolyticus in naturally contaminated seafood samples

We investigated the efficacy of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of seafood samples naturally contaminated with Vibrio parahaemolyticus. A total of 171 seafood samples enriched in alkaline peptone water (APW) were assessed by LAMP assay and conventional culture methods, which consist of a combination of APW enrichment culture and plating onto CHROMagar Vibrio and TCBS agars. Compared with V. parahaemolyticus isolation using the conventional culture test, LAMP results showed 100% (30/30) and 90.8% (128/141) sensitivity and specificity, respectively. The conventional culture test required more than 3 days to isolate and identify V. parahaemolyticus in the APW enrichment culture. In contrast, the LAMP assay was markedly faster, requiring less than 60 min from the beginning of DNA extraction to final detection of V. parahaemolyticus. In total, the LAMP assay required 17-19 h from the beginning of enrichment culture to final determination. This is the first report of the LAMP assay for rapid screening of seafood samples naturally contaminated by V. parahaemolyticus.     

Growth of Salmonella enteritidis and Salmonella typhimurium in the presence of quorum sensing signalling compounds produced by spoilage and pathogenic bacteria

The effect of acylated homoserine lactones (AHLs) and autoinducer-2 (AI-2) signalling compounds present in the cell-free culture supernatants (CFS), of Pseudomonas aeruginosa, Yersinia enterocolitica-like GTE 112, Serratia proteamaculans 00612, Y. enterocolitica CITY650 and Y. enterocolitica CITY844, on the growth of two Salmonella Enteritidis and two S. Typhimurium strains was assessed though monitoring of changes in conductance of the medium. Detection times (T_det), area and slope of conductance curves were recorded. Except for P. aeruginosa 108928, which was not found to produce AI-2, all other strains produced both AHLs and AI-2. Thereafter, aliquots (20% in the final volume) of these CFS were transferred into NZ Amine broth inoculated with ca. 10³ CFU/ml of stationary phase cultures of each Salmonella strain. While the CFS of P. aeruginosa induced a shorter detection time, i.e. acceleration of the metabolic activity, the CFS of the other microorganisms increased the detection time of Salmonella strains compared to control samples (i.e. without CFS). Results indicate that the growth of Salmonella may be affected by the presence of Quorum sensing (QS) signalling compounds and/or other novel signals existing in CFS, produced by other bacterial species and confirm the complexity of bacterial communication.