Application of multiplex PCR for monitoring colonization of pig tonsils by Yersinia enterocolitica, including biotype 1A, and Yersinia pseudotuberculosis

A multiplex PCR assay was developed for the detection and differentiation of the Yersinia enterocolitica and Yersinia pseudotuberculosis isolates in both pure bacterial cultures and pig tonsils. The assay was based on the amplification of the ail, inv, yadA, and ystB genes. The PCR products, corresponding to the ail gene and the plasmid-borne yadA gene or only one product corresponding to the ail gene, were detected in Y. enterocolitica 4 biotype isolates. All of the Y. pseudotuberculosis isolates (n=6) tested gave a positive PCR reaction for the inv gene. For all tested Y. enterocolitica 1A biotype isolates (n=31), one product corresponding to the ystB gene was observed. The multiplex PCR assay was used to detect Y. enterocolitica and Y. pseudotuberculosis strains in pig tonsil samples obtained from 80 slaughtered pigs from three different herds. The presence of at least one of the specific PCR amplification products of ail-, ystB-, yadA-, and inv-specific sequences was observed in 11 samples (13.75%). These results of the multiplex PCR assay were compared with the results of conventional, microbiological testing. Y. enterocolitica isolates were cultured from only 3 (3.75%) of the 80 pig tonsils examined. The multiplex PCR assay was shown to be an efficient tool for differentiation between the pYV plasmid-bearing Y. enterocolitica isolates, the plasmidless Y. enterocolitica isolates, the Y. enterocolitica biotype 1A isolates, and the Y. pseudotuberculosis isolates with and without the pYV plasmid in naturally contaminated pig tonsils. This indicates that this assay is useful to control food processing and track the source of contamination.     

Response surface models to predict potato tuber infection by Fusarium sambucinum from duration of wetness and temperature,and dry rot lesion expansion from storage time and temperature

Sucrose-negative Yersinia enterocolitica isolates of bioserotype 4/O:3 have been recovered for the first time. They were found in 2% of the tonsils of clinically healthy fattening pigs. These sucrose-negative Y. enterocolitica isolates could not be differentiated from Y. kristensenii isolates using API 20E; thus, they were identified using PCR and sequencing. Using pulsed-field gel electrophoresis (PFGE). NotI profiles of sucrose-negative Y. enterocolitica 4/O:3 isolates showed a high similarity to sucrose-positive Y. enterocolitica 4/O:3 isolates. This study demonstrated that sucrose-negative Y. enterocolitica 4/O:3 isolates of porcine origin can harbour virulence genes; plasmid-encoded virulence markers were found in 8 out of 11 isolates and all isolates contained chromosomal-encoded virulence markers. Thus, the pathogenicity of sucrose-negative Yersinia isolates should always be assessed.     

Yersinia pseudotuberculosis with limited genetic diversity is a common finding in tonsils of fattening pigs

A total of 425 pig tonsils, including 210 tonsils from fattening pigs and 215 from sows, from seven different abattoirs in Finland were studied for the occurrence of Yersinia pseudotuberculosis from 1999 to 2000. The mean prevalence of Y. pseudotuberculosis in fattening pig tonsils was 4%, varying from 0 to 10% between slaughterhouses. Y. pseudotuberculosis was not recovered from sow tonsils. All 30 Y. pseudotuberculosis isolates from eight pig tonsils were recovered after cold enrichment. Seventeen isolates from seven tonsils were found after cold enrichment for 14 days, followed by alkali treatment. Y. pseudotuberculosis was not isolated after direct plating, overnight enrichment, or selective enrichment. All 30 isolates belonged to bioserotype 2/0:3 and carried the virF gene in the virulence plasmid. The isolates exhibited calcium dependence and Congo red absorption. The pyrazinamidase test gave variable results. All isolates were characterized with pulsed-field gel electrophoresis (PFGE). Using SpeI, NotI, and XbaI enzymes, seven, five, and two different PFGE patterns were obtained, respectively. A total of 11 genotypes, gI to gXI, identified by a combination of the various SpeI, NotI, and XbaI profiles, were detected. Three pigs were found to carry more than one genotype. Overall, variations between PFGE patterns were small, indicating genetic homogeneity among pig strains of bioserotype 2/0:3.     

Detection, isolation and enumeration of Yersinia enterocolitica from raw pork

The methods available for the isolation of Yersinia enterocolitica from foods are generally considered to be less than optimal, and methods for estimation of numbers are lacking. Such methods are needed to understand better the significance of foodborne yersiniosis and to provide data for exposure assessment. We describe a method for the detection and enumeration of Y. enterocolitica containing the pYV virulence plasmid (YeP+) in samples from pork surfaces. The method uses a multiplex PCR targeting the ail and virF genes to detect Y. enterocolitica after incubation of surface swabs in Yersinia enrichment broth according to Ossmer. Enumeration was achieved by adapting the enrichment to a most probable number (MPN) method format. A presumptive result was available within 24 h of sample receipt, and YeP+ isolates were confirmed within four days. The presence/absence and MPN methods were evaluated in a pilot survey of 34 packs of raw pork meat purchased from retail outlets in Christchurch, New Zealand. YeP+ was detected by PCR on meat from 32% of the packs, and YeP+ isolates were obtained from 18% of the samples. YeP+ were present at numbers ranging from 0.30 to 5.42 MPN/cm(2). This improved method for the detection and enumeration of YeP+ from meat samples can be used for microbiological surveys to obtain data for assessments of consumer exposure to virulent Y. enterocolitica, and in outbreak investigations.     

Prevalence of pathogenic Yersinia enterocolitica in pigs slaughtered at a Swiss abattoir

Human yersiniosis is the third most common enteric disease after campylobacteriosis and salmonellosis in many European countries. However, epidemiological data on the prevalence of pathogenic Yersinia enterocolitica in animals and humans is insufficient. Pigs are assumed to be the main reservoir of pathogenic Y. enterocolitica because pig is so far the only animal species from which pathogenic strains have frequently been isolated. This work was conducted to study the frequency of ail-positive Y. enterocolitica in pigs slaughtered at a Swiss abattoir. In total, 212 pig tonsils were screened by real-time PCR and culture methods. The prevalence rate of ail-positive Y. enterocolitica in pigs at slaughter was 88% and 34% with PCR and culture methods, respectively. The 148 ail-positive isolates from the 72 culture-positive tonsils were bio-and serotyped. The most common bioserotype was 4/O:3 found in 96% (69/72) of the culture-positive samples. However, pig was also shown to be a reservoir for ail-positive Y. enterocolitica belonging to bioserotypes 2/O:5,27 and 2/O:9, which were detected in 8% (6/72) and 1% (1/72) of the culture-positive samples, respectively. Using PFGE with NotI, only a limited number of different patterns was found. In all, 6 genotypes were obtained when 86 isolates of bioserotype 4/O:3 from 69 samples were characterised and two genotypes (N1 and N4) dominated. The biotype 4 differs clearly from biotype 2 with PFGE. Antimicrobial resistance testing of 77 ail-positive Y. enterocolitica isolates from 72 samples studied with disc-diffusion revealed that all strains were sensitive to cefotaxime, chloramphenicol, ciprofloxacin and tetracycline, which are antimicrobial agents used for treatment of human disease. The isolates of bioserotype 2/O:5,27 differed from the isolates of bioserotypes 2/O:9 and 4/O:3 in resistance to ampicillin and amoxicillin/clavulanic acid.     

Prevalence, antibiotic susceptibility, and virulence factors of Yersinia enterocolitica and related species from ready-to-eat vegetables available in Korea

A total of 673 ready-to-eat vegetable samples were collected in Korea from 2001 to 2002 and analyzed for the presence of Yersinia spp. We analyzed biotypes, serotypes, and susceptibility to 12 antibiotics and tested for virulence genes of pathogenic Yersinia enterocolitica isolates by PCR assay. Among the samples, 27 (4.0%) were found to be contaminated with Yersinia spp. Among the 27 strains of Yersinia spp. isolates, 18 strains (66.7%) of Y. enterocolitica, 5 strains (18.5%) of Y. frederiksenii, 3 strains (11.1%) of Y. intermedia, and 1 strain (3.7%) of Y. kristensenii were identified. According to the serotypes of Y. enterocolitica isolates, O:3 (11.1%) and O:5 (11.1%) were the most predominant, followed by O:8 (5.6%) and others (72.2%). For biotypes of Y. enterocolitica isolates, 1A (77.8%) was the most predominant, followed by 3B (11.1%), 3 (5.6%), and 5A (5.6%). Also, an antibiotic susceptibility test showed that Y. enterocolitica isolates were very susceptible to the antibiotics tested but highly resistant to ampicillin (94%), cephalothin (100%), and carbenicillin (83%). PCR assays with specific primers derived from yst and ail genes of Y. enterocolitica were applied to confirm the presence of pathogenic Y. enterocolitica. Among the 18 strains of Y. enterocolitica isolates, only 3 strains (O:3/1A, UT/3B, and UT/1A isolated from Chinese cabbage, onion, and spinach, respectively) were shown to have a virulence gene.     

Isolation and characterization of Yersinia enterocolitica from swine feces recovered during the National Animal Health Monitoring System Swine 2000 study

A national study was conducted for the isolation of pathogenic Yersinia enterocolitica in pig feces in the United States as part of the National Animal Health Monitoring System's Swine 2000 study. Fecal samples collected from swine operations from September 2000 to March 2001 from 77 production sites in 15 of the top 17 swine-producing states were tested for the presence of pathogenic Y. enterocolitica. After enrichment of swine fecal samples in irgasan-ticarcillin-potassium chlorate broth, the enriched cultures were plated on cefsulodin-irgasan-novobiocin agar for isolation of presumptive Y. enterocolitica. The isolates were confirmed as pathogenic Y. enterocolitica by the fluorogenic 5' nuclease PCR assay targeting the chromosomal attachment invasion ail gene. Of 2793 fecal samples tested, 106 (3.80%) ail-positive strains of Y. enterocolitica were isolated. These 106 ail-positive isolates originated from 7 of the 15 participating states. The predominant serotype O:3 (n = 79 of 106) was distributed in five states (n = 5 of 7). Serotype O:5 (n = 27 of 106) was also found in five states (n = 5 of 7). All isolates contained the virulence plasmid and expressed virulence-associated phenotypic characteristics. These results indicate that swine in the United Stares harbor Y. enterocolitica that can potentially cause human illness.     

Prevalence of Yersinia enterocolitica in fattening pigs

The prevalence of pathogenic Yersinia enterocolitica in pig herds was monitored during six trials (at four different farrow-to-finisher farms). Samples were taken throughout the whole rearing period from birth of the piglets to the final fattening stage, and different samples were taken from these pigs during the slaughter process. Environmental samples also were evaluated to identify potential sources of on-farm infection. Y. enterocolitica was isolated using irgasan-ticarcillin-potassium chlorate broth enrichment and cefsulodin-irgasan-novobiocin agar culture. Colonies were identified using bio- and serotyping methods and by PCR assay. Pathogenic Y. enterocolitica were not isolated from fecal samples from piglets and weaners. The only fecal samples positive for Y. enterocolitica were obtained during the fattening stage. The prevalence of Y. enterocolitica in fattening pig herds ranged between 0 and 65.4%. Y. enterocolitica isolates were detected at the abattoir in 38.4% of the tonsils, in 3.8% of the ileocecal lymph nodes, on 0.3% of the carcass surfaces before chilling, and on 0% of the carcass surfaces after chilling. Almost all isolates belonged to bioserotype 4/O:3. Only one strain was identified as O:9. All isolates contained the ail gene. The yopT gene was found in 99.1% of the farm isolates but in only 76.6% of the isolates found at the abattoir from the corresponding carcasses. Although a direct link between porcine isolates and human infection has not been demonstrated, the similarity of the bioserotypes in infected pigs and humans and the presence of virulence factors in porcine isolates should encourage further studies to determine the risk of transmission of Y. enterocolitica to humans from pigs and pork products.     

Radiation resistance of virulence plasmid-containing and plasmid-less Yersinia enterocolitica

Yersinia enterocolitica, a foodborne pathogen, can be eliminated from meat by ionizing radiation. Y. enterocolitica sometimes contains a 70-kb virulence plasmid that encodes genes for a type III secretion channel and host immune suppression factors. The radiation resistance of virulence plasmid-containing and plasmid-less Y. enterocolitica was determined. Four Y. enterocolitica serotypes containing (i) the large virulence plasmid, and (ii) their plasmid-less derivatives were inoculated into raw ground pork, which was then vacuum packed and irradiated at 4 degrees C to doses of 0.2, 0.4, 0.6, 0.8, and 1.0 kGy. The D10-value, the radiation dose required to reduce the number of viable Y. enterocolitica by 90%, was not dependent on the presence or absence of the virulence plasmid, but it did differ among the four Y. enterocolitica serotypes.     

Genotypic and phenotypic diversity of Lactococcus lactis isolates from Batzos, a Greek PDO raw goat milk cheese

The genotypic and phenotypic variability of 40 Lactococcus lactis isolates obtained from three cheese-making trials of Batzos cheese made one in each, winter, spring and summer was investigated. RAPD-PCR, plasmid profiling and PFGE were used to study the genetic variability and distinguish closely related isolates. Results showed a high degree of heterogeneity among strains. According to PFGE data, all strains except one were clustered together (at a similarity level of approximately 50%) with the L. lactis subsp. lactis reference strain and eleven groups of isolates consisting of 2-8 strains each were distinguished. Plasmid profiling results revealed that there were eight isolates lacking plasmids and nine having unique plasmids. Twenty-three isolates were allocated into six groups. There was an interesting similarity between the plasmid profiling groups and those formed according to PFGE. Clustering of strains according to RAPD-PCR was in agreement with results obtained by both plasmid profiling and PFGE for the majority of the strains. In addition, results obtained by molecular methods indicate a grouping of most of the strains according to the season of cheese production. All strains inhibited the growth of Escherichia coli O157:H7. Their ability to affect the growth of Yersinia enterocolitica, Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes and Enterococcus faecalis was strain dependent. In 42.5% of the isolates high acidifying ability in milk after 24 h was recorded and these were isolates, mainly, from fresh cheese. The 75% of the isolates from winter cheese exhibited higher Lys- than Leu-aminopeptidase activity while the approximately 67% of the isolates from summer cheese showed higher Leu- than Lys-aminopeptidase activity. Their caseinolytic activity after growth in milk for 24 h was significant with preference for alpha(s)-casein degradation. The majority (90%) of the strains formed methanethiol from methionine and this ability was strain dependent. These results suggest that among the wild lactococcal population from Batzos cheese there are interesting strains appropriate to be used as starters for the dairy industry.     

Yersinia enterocolitica in milk and dairy products

Yersinia enterocolitica was first recognized during the 1960's as an important human enteropathogen. The species as later redefined includes both pathogenic and nonpathogenic forms. Pathogenic strains that retain the virulence plasmid can be identified in several animal models and four indirect tests (calcium dependency, autoagglutination, Congo red uptake, serological detection of outer membrane antigen) and by tissue culture assay, serotype, and biotype. Y. enterocolitica and related bacteria have frequently been isolated from raw milk, but none of the isolates, with the possible exception of serotype 05,27, are recognizable as pathogens. Under normal circumstances Y. enterocolitica does not survive pasteurization. If introduced into pasteurized milk, it can grow well at refrigeration temperatures. Two outbreaks of yersiniosis have occurred that involved pasteurized milk. Pigs, which frequently carry pathogenic Y. enterocolitica in their throat, were the probable source in one of these outbreaks. The most rapid enrichment procedure available for isolation of Y. enterocolitica requires 6 d. No isolation method is available for selective isolation of pathogenic Y. enterocolitica in the presence of related bacteria common in milk and other foods.     

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.     

Detection of chromosomal and plasmid--encoded virulence determinants in Yersinia enterocolitica and other Yersinia spp. isolated from food animals in Greece

The distribution of Yersinia strains in animal reservoirs was examined in 835 food animals (pigs, chickens, sheep, cows) from different Greek departments (Attica, Fthiotida, Viotia and Evia) over a one year period. The isolated strains were characterized with respect to the presence of chromosomal (yst) and plasmid-encoded virulence determinants (virF, yadA) and their antimicrobial susceptibility was tested.In total, Yersiniaspp. were obtained from 9.94% of the 835 food animals at slaughter that were sampled in this study. There was no statistically significant seasonal distribution, nor was any significant departmental distribution observed. From the 83 isolated Yersinia strains, 76 (91,57%) belonged to Y. enterocolitica (58 were of serotype O:3/biotype 4 and 18 strains were non O:3, non O:9), 3 belonged to Y. pseudotuberculosis, 2 to Y. kristensenii and 2 to Y. intermedia. Y. enterocolitica O:3/4 was mainly isolated from the pigs, while Y. enterocolitica non O:3, non O:9 was from the chickens. The strains were grouped into 5 genotypes, with respect to the presence or absence of the virulence genes. A significant predominance of genotype V, the one carrying all the three virulence genes, was observed in the strains isolated from the pigs. Complete susceptibility to most of the 3rd and to the 4th generation cephalosporins and to ciprofloxacin, was observed among the isolates. Remarkable was the association between the presence of each virulence gene separately and resistance to some antimicrobials, a matter of further investigation.     

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.     

Comparison of Selected Yersinia enterocolitica Indicator Tests for Potential Virulence

Three recently developed tests used to indicate virulence were performed on 34 strains of Yersinia enterocolitica. Agreement in virulence or nonvirulence prediction was found in (a) 29 of 31 strains when tested for calcium growth dependency at 35°C; (b) all 34 strains when tested for the ability to autoagglutinate in tissue culture medium at 35°C; and (c) among all 34 strains when tested for the presence of a 40–48 Mdal plasmid using a modification of the Hansen and Olsen method for plasmid analysis. The autoagglutination assay is recommended for testing potential pathogenicity of food-borne isolates. There are probably several virulence-determining factors for Y. enterocolitica, and no one in-vitro test will suffice. The analytical procedure described herein can be used to detect plasmids ranging in size from approximately 2 × 10⁶ to 3 × 10⁸ daltons.     

牛奶源金黄色葡萄球菌血清型、毒力基因及PFGE分型

目的：研究生鲜牛奶中金黄色葡萄球菌分离、荚膜多糖血清型分布、毒力基因携带及脉冲场凝胶电泳（pulsed field gel electrophoresis,PFGE）分型情况。方法：从河南省4?个地区奶牛养殖场采集生鲜牛奶样品按照国标法进行分离,扩增耐热核酸酶基因nuc鉴定金黄色葡萄球菌,利用聚合酶链式反应方法测定荚膜多糖血清型和毒力基因携带情况,采用PFGE分析菌株间的相关性和遗传关系。结果：从350?份生鲜牛奶样品中分离鉴定到80?株金黄色葡萄球菌,分离率为22.86%。荚膜多糖血清型测定发现,cap5（60%）是流行血清型。从这些阳性菌株中,发现有62?株（77.5%）携带有毒力基因,毒力基因set、hlb、hld、lukED、ebp、clfA和clfB,检出率分别为40.00%、51.25%、57.50%、60.00%、58.75%、57.50%和58.75%。此外,47?株（58.75%）菌携带不少于6?个毒力基因,流行的毒力基因谱型为set-hla-hlb-hld-lukED-cna-ebp-clfA-clfB。PFGE结果显示,获得72?株菌的PFGE图谱,按90%的相似性可分为12?个簇和46?种PFGE型。D簇（3?种PFGE型）、G簇（3?种PFGE型）和J簇（5?种PFGE型）菌株中均检出一定基因类型的毒力基因,表明河南地区生鲜牛奶中金黄色葡萄球菌毒力基因广泛存在于多种PFGE型别中。结论：生鲜牛奶均有一定程度的金黄色葡萄球菌污染,多数菌株携带毒力基因,且毒力基因的类型较为复杂,这对消费这些牛奶的人群构成潜在的健康威胁。PFGE分型菌株主要以克隆形式进行传播,且克隆型具有多样性和差异性,故临床应加强生奶及乳品血清型、毒力基因检测及分子分型研究。     

Interlaboratory random amplified polymorphic DNA typing of Yersinia enterocolitica and Y. enterocolitica-like bacteria

A random amplified polymorphic DNA (RAPD) protocol was developed for interlaboratory use to discriminate food-borne Yersinia enterocolitica O:3 from other serogroups of Y. enterocolitica and from Y. enterocolitica-like species. Factors that were studied regarding the RAPD performance were choice of primers and concentration of PCR reagents (template DNA, MgCl(2), primer and Taq DNA polymerase). A factorial design experiment was performed to identify the optimal concentrations of the PCR reagents. The experiment showed that the concentration of the PCR reagents tested significantly affected the number of distinct RAPD products. The RAPD protocol developed was evaluated regarding its discrimination ability using 70 different Yersinia strains. Cluster analysis of the RAPD patterns obtained revealed three main groups representing (i) Y. pseudotuberculosis, (ii) Y. enterocolitica and (iii) Y. kristensenii, Y. frederiksenii, Y. intermedia and Y. ruckeri. Within the Y. enterocolitica group, the European serovar (O:3) and the North American serovar (O:8) could be clearly separated from each other. All Y. enterocolitica O:3 strains were found in one cluster which could be further divided into two subclusters, representing the geographical origin of the isolates. Thus, one of the subclusters contained Y. enterocolitica O:3 strains originating from Sweden, Finland and Norway, while Danish and English O:3 strains were found in another subcluster together with O:9 and O:5,27 strains. The repeatability (intralaboratory) and reproducibility (interlaboratory) of the RAPD protocol were tested using 15 Yersinia strains representing different RAPD patterns. The intralaboratory and the interlaboratory studies gave similarity coefficients of the same magnitude (generally >70%) for the individual strains. In the present study, it was shown that interreproducible RAPD results could be achieved by appropriate optimisation of the RAPD protocol. Furthermore, the study reflects the heterogeneous genetic diversity of the Y. enterocolitica species.     

Detection, semiquantitative enumeration, and antimicrobial susceptibility of Yersinia enterocolitica in pork and chicken meats in Italy

Yersinia enterocolitica is recognized as an etiological agent of gastroenteritis, lymphadenitis, and chronic sequelae. During 2006 and 2007, 205 samples (125 pork and 80 chicken meats) were collected in Italy and tested for detection and most-probable-number (MPN) enumeration of Y. enterocolitica organisms. The microorganism was isolated from 45 samples (21.9%): 19 (15.2%) pork samples and 26 (32.5%) chicken samples. Y. enterocolitica MPN contamination levels were low, ranging from 0.30 to 1.50/g. Most (94.4%) Y. enterocolitica strains were biotype 1A (serotypes O:3; O:5; O:6,30; O:6,30-6,31; O:7,8-8-8,19; O:8; O:9; O:25,35; O:36; and O nontypeable), and 5.6% of the isolates were bioserotype 2/O:9. All isolates were tested for yadA, ail, inv, ystA, and ystB virulence sequences. The yadA gene was detected in two strains (3.7%) isolated from chicken samples: one Y. enterocolitica 2/O:9 yadA+ ail+ ystA+, and one Y. enterocolitica 1A/O:7,8-8-8,19 yadA+ inv+ ystB+. Two (3.7%) 2/O:9 strains, isolated from pork products, were ail+ ystA+. Most biotype 1A strains were ystB+ (84.3%) and inv+ (39.2%). All strains were sensitive to cefotaxime, ciprofloxacin, chloramphenicol, nalidixic acid, streptomycin, sulfonamide, tetracycline, trimethoprim, and trimethoprim-sulfamethoxazole. Resistance to gentamicin and aztreonam was observed in 1.9% of the isolates. High levels of resistance were detected toward amoxicillin-clavulanic acid (27.8%), ampicillin (75.9%), and erythromycin (100%). The authors hypothesize that Y. enterocolitica pathogenic biotypes are rather uncommon in foods when compared with their isolation rates from animal sources and that chicken meat could be contaminated as well as pig meat and its derived products.     

台州市食源性沙门菌耐药性、毒力因子及分子分型研究

目的 了解台州市食品中分离的沙门菌耐药性、毒力因子以及基因分型情况,建立食源性沙门菌的分子特征本底信息,为食源性疾病的防治提供技术支撑.方法 对近几年从食品中分离的22株沙门菌进行12种抗生素药敏试验、10种毒力基因PCR检测、脉冲电场凝胶电泳(PFGE)基因分型,用BioNumerics 5.0软件对分型数据进行聚类分析.结果 22株食源性沙门菌的总耐药率为59.1％,耐药率居前三位的抗生素分别是复方新诺明(36％)、四环素(27％)、萘啶酸(27％);所有菌株均检出6种以上毒力因子,1株肠炎沙门菌存在毒力岛、质粒及噬菌体等多种毒力因子;PFGE分型共得到21个条带,可分为5个基因型别,包括18种指纹图谱,各基因型别间同源性小于70％.结论 台州市食源性沙门菌存在致病风险,建立的指纹图谱数据库可为食源性疾病的防治提供技术支持.     

Prevalence of virulent Yersinia enterocolitica in bulk raw milk and retail cheese in northern-west of Iran

To determine the prevalence of virulent Yersinia enterocolitica, 554 samples consisting of 354 bulk raw milks and 200 traditional cheeses were collected from different parts of Eastern-Azerbaijan province, during a 23-month period from 2008 to 2010. The occurrence of virulent strains of Y. enterocolitica in samples enriched in peptone sorbitol bile broth (PSBB) was evaluated via the detection of attachment invasion locus (ail) gene by PCR. The viability of virulent Y. enterocolitica in the PCR-positive samples was tested using conventional culture method and the isolates were confirmed by the second-phase ail-PCR. According to the results, 8.66% of total samples including 7.62% of bulk raw milks and 10.5% of raw milk cheeses were found ail-positive by PCR method; subsequently Y. enterocolitica was isolated by the culture method and confirmed by the second phase ail-PCR in 2.88% of total samples including 2.26% of raw milks and 4% of cheese samples. It was concluded that, a sample enrichment followed by ail-PCR was more sensitive and robust to detect and distinguish the virulent strains of Y. enterocolitica compared to the conventional culture method.