Isolation of Yersinia enterocolitica from foods.

Many selective enrichment and plating media for the isolation of Yersinia enterocolitica from foods are described. However, at present no single isolation procedure is available for the recovery of all pathogenic strains of Yersinia enterocolitica. Cold enrichment in phosphate-buffered saline plus 1% sorbitol and 0.15% bile salts (PBSSB) and two-step enrichment with tryptone soy broth (TSB) and bile oxalate sorbose (BOS) broth are very efficient methods for the recovery of a wide spectrum of serotypes of Y. enterocolitica. Enrichment in irgasan ticarcillin chlorate (ITC) broth was found to be the most efficient method for the recovery of strains of serotype 0:3, which is the most common clinical serotype of Y. enterocolitica in Europe. Post-enrichment alkali treatment often results in higher isolation rates. Cefsulodin irgasan novobiocin (CIN) agar and Salmonella-Shigella deoxycholate calcium chloride (SSDC) agar are the most commonly used plating media. For the recovery of serotype 0:8 strains, the common clinical isolates in North America, enrichment in BOS and plating on CIN seems the most efficient procedure. Selection of the proper enrichment procedure will depend on the bio/serotypes of Yersinia spp. sought and on the type of food to be examined. The use of more than one medium for both enrichment and plating will result in higher recovery rates of Yersinia spp. from foods. Parallel use of the following two isolation procedures is recommended. (1) Enrichment in ITC for 2 days at 24 degrees C; plating on SSDC agar (2 days at 30 degrees C). (2) Pre-enrichment in TSB for 1 day at 24 degrees C; enrichment in BOS for 5 days at 24 degrees C; alkali treatment (mixing 0.5 ml enriched broth with 4.5 ml of 0.5% KOH in 0.5% NaCl for 5 s); plating on CIN agar (2 days at 24 degrees C).     

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.     

Isolation of Potentially Virulent Yersinia enterocolitica from Variety Meats

Sixty-three samples of variety meats were examined for the presence of Yersinia enterocolitica. Surface swab samples in a modified selenite enrichment broth were incubated at 23°C for 2 and 3 days and then streaked onto MacConkey agar with Tween 80 or refrigerated bismuth sulfite agar, with and without a potassium hydroxide preplating treatment. The organism was isolated from 2 of 23 porcine throat swabs but was not isolated from 40 bovine tongue, kidney, heart, or liver swabs. The isolates from the throat swabs were classified into Nilehn and Wauters biotypes 3 or 4. One porcine isolate was of serotype 0:19, while another isolate from the second Y. enterocolitica-bearing porcine throat swab was nontypeable. The nontypeable isolates autoagglutlnated in tissue culture when grown overnight at 35°C, suggesting that these isolates are potentially virulent. The results of this study support the contention of European researchers that swine may be a significant source of Y. enterocolitica involved in human infections.     

Isolation of Yersinia enterocolitica from raw cow milk in Argentina

A survey was carried out to determine the presence of Yersinia enterocolitica in raw cow milk from producers in Buenos Aires, Argentina. From March to June 1984, 18 pooled- and 93 individual-producer samples were examined by two enrichment procedures. Y. enterocolitica was isolated from 6 samples (5.5%). Eleven samples (9.9%) yielded Yersinia frederiksenii and 4 (3.6%) Yersinia intermedia. All the isolates of Y. enterocolitica belonged to biotype 1 of Wauters. Two serotypes, 0:5 and 0:10,34 were identified. The phage types were divided into Xo and Xz. The strains were avirulent by calcium dependence, autoagglutination and mouse i.p. injection tests.     

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.     

Slaughter pigs and pork as a source of human pathogenic Yersinia enterocolitica.

Pathogenic Yersinia enterocolitica strains (serogroups 0:3;0:9 and 0:5.27) were isolated from 36 (42%) of 86 porcine tonsils, 8 (20%) of 40 tongues, 17 (17%) of 100 rectal swabs and from 4 (1%) of 400 pork samples. Pathogenic Yersinia strains were not isolated from samples of 210 pig carcasses and from 20 samples of porcine head meat. These results confirm that pigs are an important reservoir of pathogenic Y. enterocolitica. However, contamination of carcasses during the slaughtering process with Yersinia from either faecal material or from the tonsillary region does not seem to occur frequently and this may also explain the low contamination rate of pathogenic Y. enterocolitica found for pork. For the isolation of pathogenic Y. enterocolitica strains from foods, enrichment in irgasan-ticarcillin-chlorate broth (ITC) and isolation on SS-deoxycholate-calcium agar (SSDC) is recommended.     

Isolation of Yersinia enterocolitica and Related Species from Red Meat and Milk

A total of 150 samples, 50 each of beef, lamb and pork from 10 local retail stores in the Brisbane metropolitan area and 50 pasteurized and 150 raw bovine bulk milk tank samples obtained from Queensland United Foods (QUF), were examined for the presence of Yersinia spp. over 1 yr. Two isolation protocols were used to recover the organism with subsequent biochemical and serological identification. A total of 114 isolates, consisting of Y. enterocolitica (23,12,15,40), Y. intermedia (0,3,5,1), Y. frederiksenii (4,1,0,10) were obtained from beef, lamb, pork, and milk, respectively. No pasteurized milk samples were positive for the organism. None of the isolated strains was found to harbor the virulence plasmid as indicated by the crystal violet-binding assay.     

Occurrence of pathogenic Yersinia enterocolitica in Norwegian pork products determined by a PCR method and a traditional culturing method

From October 1997 to April 1998, a survey was conducted to assess the occurrence of pathogenic Yersinia enterocolitica in Norwegian pork products, using a traditional culturing method and a PCR assay. A total of 300 pork samples was examined. Five slaughterhouses in the Norwegian Meat Cooperative were represented with 249 samples and another 51 samples were obtained from retail outlets in the city of Oslo. Using the NMKL method, Y. enterocolitica 0:3 was isolated from six (2%) of the samples, while the PCR method indicated presence of pathogenic Y. enterocolitica in 50 (17%) of the samples. The results indicate that a reduction has occurred in the prevalence of pathogenic Y. enterocolitica in Norwegian pork products, as compared to a previous Norwegian study conducted in 1988-1989. The study also highlights the need for further development and improvement of methods applied for the detection of pathogenic Y. enterocolitica in foods.     

Antimicrobial resistance of Yersinia enterocolitica strains from human patients, pigs and retail pork in Switzerland

To obtain basic data for future resistance monitoring programs, 386 Yersinia enterocolitica strains from human patients, raw retail pork and pig feces were tested for their susceptibilities to 16 antimicrobial agents and two antimicrobial growth promoters (carbadox and olaquindox). No strains were resistant to ceftriaxone, cefuroxime, ciprofloxacine, gentamicin, kanamycin, neomycin or polymyxin. Although in Switzerland carbadox and olaquindox were used as growth promoters for pigs for over 25 years, all strains were susceptible to them. In contrast, there were high levels of resistance to ampicillin, cefalothin and amoxicillin/clavulanic acid. Less than 10% of clinical isolates and strains from pig feces were resistant to streptomycin, sulfonamide, trimethoprim/sulfamethoxazole, tetracyclin, trimethoprim and chloramphenicol, but strains from retail pork were all susceptible to these antimicrobial agents. This finding suggested that pork is probably not a major source of Y. enterocolitica that cause human infections in Switzerland. A difference between clinical isolates and strains from pork was also shown by serotyping. Clinical isolates frequently belonged to the O3 and O9 groups whereas these two serotypes were not found in strains from pork. Resistance to multiple antimicrobial agents was rare. When examined by pulsed field gel electrophoresis (PFGE), two strains of fecal origin with an identical pattern of resistance to six antimicrobial agents were shown to be unrelated. Of four clinical isolates with resistances to five antimicrobial agents, two were of the same pulsotype. Retrospectively, it was found that these strains came from two members of the same household and thus represented a mini-outbreak.     

小肠结肠炎耶尔森菌在猪肉中失活/存活模型的初步研究

采用预测微生物学的基本方法和程序,研究小肠结肠炎耶尔森菌在特定条件下的生长规律,并用CurveExpertl.38软件作为辅助工具,从中选取了Linear模型对实验数据进行拟合.初步建立了肉汤中小肠结肠炎耶尔森菌55、60℃的热失活模型,以及猪肉中小肠结肠炎耶尔森菌的速冻失活模型和冷冻存活模型.     

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.     

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.     

Expression of major cold shock proteins and genes by Yersinia enterocolitica in synthetic medium and foods

Yersinia enterocolitica is a psychrotrophic foodborne pathogen that has been implicated in outbreaks of foodborne illness involving cold-stored foods, especially milk and pork. A major mechanism bacteria use to adapt to cold is expression of cold shock proteins. The objective of this research was to study the expression of major cold shock proteins of Y. enterocolitica in Luria-Bertani (LB) broth, milk, and pork following a temperature downshift from 30 to 4 degrees C. Y. enterocolitica was inoculated into 10 ml of LB broth, sterile skim milk, or pork, and the samples were stored at 4 degrees C (cold shock) or 30 degrees C (control) for 0, 4, 8, 12, and 24 h. At each sampling time, total protein and total RNA were extracted from Y. enterocolitica harvested from LB broth, milk, and pork and subjected to two-dimensional gel electrophoresis and dot blot analysis. Two major cold shock proteins (CspA1 and CspA2) of approximately 7 kDa and their genes were expressed by Y. enterocolitica following cold shock. However, the CspA1 and CspA2 proteins were not expressed by Y. enterocolitica at 30 degrees C. Y. enterocolitica CspA1 and CspA2 were observed as early as 2 h of cold shock in cultures from LB broth and milk and at 8 h of cold shock in cultures from pork.     

新等温扩增技术检测小肠结肠炎耶尔森氏菌

应用新型等温扩增检测技术——交叉引物等温扩增结合免疫金标试纸条建立检测小肠结肠炎耶尔森氏菌的方法。针对小肠结肠耶尔森氏菌16-23S rDNA间区序列设计特异性引物及探针,用54株小肠结肠炎耶尔森氏菌及相近株细菌进行特异性试验;通过纯菌液计数、样品中添菌检测进行灵敏度验证;对677份食品用传统生化国标法进行比较检测试验。建立方法具有较好特异性;增菌液检测灵敏度为10~1cfu/mL,当每25g样品中有10~0cfu菌时经增菌步骤后即可检出,样品检测同传统检测结果比较大致相符,没有漏检,假阳性率较低。建立的新型恒温检测方法可用于食品中小肠结肠炎耶尔森氏菌初筛检测。     

致病性小肠结肠炎耶尔森氏菌分离鉴定及三重PCR检测方法的建立

为鉴定导致患病鲫鱼肛门红肿、腹部有出血点症状的病原菌,并建立一种快速检测该病原菌的方法.本研究从患病鲫鱼中分离了病原菌,采用形态学、理化特性分析及16SrDNA序列分析方法鉴定菌株.采用PCR扩增法检测该菌毒力基因,琼脂纸片扩散法检测该菌株的耐药性,致病性能验证试验检测其致病性.针对该菌ail基因、inv基因和intB...     

Enrichment, isolation, and virulence of freeze-stressed plasmid-bearing virulent strains of Yersinia enterocolitica on pork

The influence of freeze stress at -20 degrees C on the enrichment, isolation, detection, presence of virulence plasmid, and expression of virulence of plasmid-bearing Yersinia enterocolitica (YEP+) inoculated on pork chop medallions was assessed. Pork chop medallions (10 cm2) artificially contaminated with 10, 1, and 0.5 CFU/cm2 of YEP+ strains (serotype O:3) were placed in sterile petri dishes at -20 degrees C for 24 h. The medallions were swabbed when frozen, after thawing at room temperature for 1.5 h and after thawing at 4 degrees C for 18 h. Swabs were enriched and YEP+ were detected and isolated using the Congo red-binding and low-calcium-response assays. The YEP+ were isolated under all conditions on pork chop medallions inoculated with 10 CFU/cm2 and at a level of 1 CFU/cm2 when thawed at room temperature and at 4 degrees C but not from frozen pork chop medallions. The YEP+ were not isolated from pork chop medallions inoculated with 0.5 CFU/cm2 and then frozen, whereas YEP+ were recovered when inoculated at this level from pork chop medallions not subjected to freezing. Virulence of the strains isolated from frozen pork chop medallions was confirmed by PCR and the expression of plasmid-associated phenotypes. These results indicate that YEP+ subjected to freezing on pork are potentially capable of causing foodborne illness and that freezing is not a substitute for safe handling and proper cooking of pork.     

Listeria species in raw and ready-to-eat foods from restaurants

From September 1999 to March 2000, meat (pork, beef, and chicken), fish (salmon, hake, and sole), vegetable (lettuce and spinach), and Spanish potato omelette samples obtained at restaurants were collected and tested for the occurrence of Listeria spp. Listeria monocytogenes was isolated from 3 (2.9%) out of 103 studied samples. Other species isolated were Listeria grayi (13.6%), Listeria innocua (1.9%), Listeria ivanovii (5.8%), Listeria seeligeri (3.9%), and Listeria welshimeri (1.9%). Listeria was neither isolated from beef nor any type of fish.     

一起引起食源性疾病的小肠结肠炎耶尔森菌分离菌株的检测分析

目的 了解一株引起婴幼儿腹泻的食源性疾病的小肠结肠炎耶尔森菌的血清型、毒力基因携带和耐药性.方法 通过血清凝集法、改良K-B纸片法及PCR法,对分离菌株进行血清分型、药敏试验、毒力基因(ail,ystB,virF)及0:3血清型菌株特异性脂多糖O-侧链的生物合成基因,rfbc的检测.结果 本菌株0:3血清凝集试验阳性,带有毒力基因ail、virF及0:3血清型菌株特异性鉴别基因rfbc,而毒力基因yatB阴性;该菌株已对头孢唑林、氨苄西林和复方新诺明产生耐药,但对其他头孢类、碳青霉烯类、氨基糖苷类等药物敏感.结论 引起食源性疾病的病原菌是0:3血清型且带有ail、virF毒力基因的致病性非1A型小肠结肠炎耶尔森菌.     

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.     

Evaluation of a norovirus detection methodology for ready-to-eat foods

Despite recent norovirus (NoV) foodborne outbreaks related to consumption of ready-to-eat (RTE) foods, a standardized assay to detect NoV in these foods is not available yet. Therefore, the robustness of a methodology for NoV detection in RTE foods was evaluated. The NoV detection methodology consisted of direct RNA extraction with an eventual concentration step, followed by RNA purification and a multiplex RT-qPCR assay for the detection of GI and GII NoV and the murine norovirus-1 (MNV-1), the latter used as process control. The direct RNA extraction method made use of the guanidine-isothiocyanate containing reagent (Tri-reagent?, Ambion) to extract viral RNA from the food sample (basic protocol called TriShort), followed by an eventual concentration step using organic solvents (extended protocol called TriConc). To evaluate the robustness of the NoV detection method, the influence of (1) the NoV inoculum level and (2) different food types on the recovery of NoV from RTE foods was investigated. Simultaneously, the effect of two RNA purification methods (manual RNeasy minikit (Qiagen) and automated NucliSens EasyMAG (BioMérieux)) on the recovery of NoV from these foods was examined. Finally, MNV-1 was evaluated as process control. First of all, high level GI and GII NoV inocula (~10? NoV genomic copies/10 g) could be recovered from penne salad samples (10 g) in at least 4 out of 6 PCRs, while low level GI and GII NoV inocula (~10? NoV genomic copies/10 g) could be recovered from this food product in maximally 3 out 6 PCRs, showing a significant influence of the NoV inoculum level on its recovery. Secondly, low level GI and GII NoV inocula (10? NoV genomic copies/10 g) were spiked onto 22 ready-to-eat food samples (10 g) classified in three categories (soups, deli sandwiches and composite meals). The GI and GII NoV inocula could be recovered from 20 of the 22 samples. The TriConc protocol provided better recoveries of GI and GII NoV for soups while the TriShort protocol yielded better results for the recovery of GII NoV from composite meals. NoV recovery from deli sandwiches was problematic using either protocol. Thirdly, the simultaneous comparison of two RNA purification protocols demonstrated that automated RNA purification performed equally or better compared to manual RNA extraction. Finally, MNV-1 was successfully evaluated as process control when detecting NoV in RTE foods using this detection methodology. In conclusion, the evaluated NoV detection method was capable of detecting NoV in RTE foods, although recoveries were influenced by the inoculum level and by the food type.