Detection Of Pathogenic Yersinia Enterocolitica Research Articles

A lateral flow immunochromatographic strip based on RPA and quantum dot nanobeads for rapid identification of pathogenic Yersinia enterocolitica

To detect pathogenic Yersinia enterocolitica in meat, a lateral flow immunochromatographic strip targeting on virulence gene ail was developed, which was based on recombinase polymerase amplification (RPA) and quantum dot nanobeads (QDNBs). Under optimal conditions, the minimum detection limit of the strip was 9.6 × 103 CFU/mL, with a standard curve equation of y = 0.144x - 0.5098 (R2 = 0.9863), and the detection times including the RPA reaction at hand temperature (35–36.5 °C), could be controlled in 10 min. When testing spiked sample, the recoveries (%) of Yersinia enterocolitica at 9.6 × 104 CFU/mL were 44.27 ± 2.08 and 45.31 ± 3.12 in beef and pork samples and was 107.55 ± 1.30 in chicken samples. When testing 15 actual samples, only one pork sample was tested positive, consistent with polymerase chain reaction (PCR) results. The strips are body temperature-triggered and rapidly visualized, suitable for rapid field detection of pathogenic Yersinia enterocolitica.

Establishment of a Real-Time Recombinase Polymerase Amplification for Rapid Detection of Pathogenic Yersinia enterocolitica

Yersinia enterocolitica is a zoonotic proto-microbe that is widespread throughout the world, causes self-limiting diseases in humans or animals and even leads to sepsis and death in patients with severe cases. In this study, a real-time recombinase polymerase amplification (RPA) assay for pathogenic Y. enterocolitica was established based on the ail gene. The results showed that the RPA detection for Y. enterocolitica could be completed within 20 min at an isothermal temperature of 38 °C by optimizing the conditions in the primers and Exo probe. Moreover, the sensitivity of the current RT-RPA was 10-4 ng/μL, and the study found that the assay was negative in the application of the genomic DNA of other pathogens. These suggest the establishment of a rapid and sensitive real-time RPA method for the detection of pathogenic Y. enterocolitica, which can provide new understandings for the early diagnosis of the pathogens.

Combined molecular biological and microbiological detection of pathogenic Yersinia enterocolitica in spiced ground pork, meat for production of ground pork and raw sausages

In dieser Studie wurde uber einen funfjahrigen Zeitraum (2013–2017) das Vorkommen von humanpathogenen Yersinia enterocolitica in gewurztem Schweinehackfleisch, Schweinefleisch zur Hackfleischherstellung und streichfahigen, kurz gereiften Rohwursten in Sachsen untersucht. Der Erreger konnte in 11,5% der zum Rohverzehr bestimmten Hackfleischerzeugnisse (n = 2.145) nachgewiesen werden. Rohware zur Hackfleischherstellung (n = 62) erbrachte eine Nachweisquote von 22,6%. Auch in 1,9% der Zwiebelmettwurste (n = 104) wurde Yersinia enterocolitica detektiert. Bei der Verteilung der Biovar/Serovar-Konstellationen entfielen 84,8% auf 4/O:3; 7,2% auf 2/O:9; 5,3% auf 2/O:57, 27 und 2,7% auf 3/O:3. Die hohen Nachweisquoten zeigen, dass die Krankheitserreger in verzehrfertigen Schweinefleischprodukten in Sachsen haufiger vorkommen als bisher angenommen. Neben der Ermittlung der Pravalenz wird gezeigt, dass die verwendete Methode geeignet ist, um Lebensmittel mit verhaltnismasigem Aufwand auf humanpathogene Varianten zu untersuchen. Es werden weiterhin wirksame Masnahmen benotigt, um einen Eintrag der Yersinien in die Lebensmittelkette zu vermeiden.

Validation of EN ISO method 10273 - Detection of pathogenic Yersinia enterocolitica in foods

EN ISO 10273 method for the detection of pathogenic Yersinia enterocolitica in foods was validated in the project Mandate M/381 funded by European Commission. A total of 14 laboratories from five European countries participated in the interlaboratory study (ILS) organized during 2013 and 2014. Before the ILS, the method was revised by an international group of experts and the performance of the revised method was assessed in an ILS study. The results are published as a part of the standard EN ISO 10273 revision. The study included three rounds with different sample types; raw milk, iceberg lettuce and minced meat, inoculated with a low and high level of pathogenic Y. enterocolitica strains representing major pathogenic bioserotypes 4/O:3 and 2/O:9. The homogeneity and stability of the samples were verified before dispatching them to the laboratories. The results demonstrated the method sensitivity of 96% in raw milk, 97% in minced meat, and 98% in lettuce at high inoculation level of pathogenic Y. enterocolitica. The specificity was 100% in raw milk, 96% in minced meat, and 98% in lettuce. The level of detection, LOD50, varied between study rounds, being 9.4 CFU/25 ml in raw milk, 9.9 CFU/25 g in minced meat and 63 CFU/25 g in lettuce samples. During the study, confirmation by using real-time PCR method ISO/TS 18867 together with pyrazinamidase testing was also validated, as alternative to conventional biochemical confirmation. When comparing different isolation steps used in the revised method during the study rounds, PSB enrichment and plating on CIN after alkaline (KOH) treatment showed the highest sensitivity (52–92%) in raw milk and minced meat samples. In lettuce samples, however, ITC with KOH treatment before plating on CIN showed higher sensitivity (64% at low level; 82% at high level) than plating on CIN from PSB with KOH treatment (44% at low level; 74% at high level). Statistical analysis of different isolation steps supported the use of two enrichment media, PSB and ITC, in the revised method. Recovery of pathogenic Y. enterocolitica on CIN was most efficient after KOH treatment and, based on the analysis, plating on CIN agar without KOH treatment could be left as optional procedure in the method.

Detection of Pathogenic Yersinia Enterocolitica in Slaughtered Pigs by Cultural Methods and Real-Time Polymerase Chain Reaction.

Healthy pigs carrying pathogenic to human Yersinia enterocolitica strains are the main source of entry into slaughterhouse, where cross-contamination of carcasses can happen. The aim of this work was to determine Y. enterocolitica prevalence in slaughtered pigs, investigating the presence of carriers in relation to carcass contamination. A total of 132 pig samples (tonsils, mesenteric lymph nodes, colon content, carcass surface) were collected from 4 Sardinian slaughterhouses. All the samples were examined by the ISO 10273:2003 method, and the prevalence was also determined by direct plating on CIN Agar. Moreover, to detect the ail positive Y. enterocolitica strains in enrichment broths and isolates a real-time polymerase chain reaction (PCR) was applied. Y. enterocolitica prevalence was 19% with direct plating and 12% with enrichment methods. Carcass surfaces and tonsils prevalence was 5.30% by direct plating, and 5.3% and 2.2%, respectively, by enrichment method. Tonsil samples showed an average contamination level of 3.2×103 CFU/g, while the mean value on carcass was 8.7×102 CFU/g. An overall prevalence of 9.8% of ail positive Y. enterocolitica broths was detected by RT-PCR, that found a higher prevalence in tonsils (7.5%) with respect to cultural methods, confirming the greater sensitivity of this technique when applied for tonsils and faeces samples. The results show a relatively low pathogenic Y. enterocolitica prevalence in pigs slaughtered in Sardinia. Good hygiene measures should be applied at slaughterhouse in order to prevent the entry of carriers and control carcass contamination.

A Selective Chromogenic Plate, YECA, for the Detection of Pathogenic Yersinia enterocolitica: Specificity, Sensitivity, and Capacity to Detect Pathogenic Y. enterocolitica from Pig Tonsils.

A new selective chromogenic plate, YECA, was tested for its specificity, sensitivity, and accuracy to detect pathogenic Y. enterocolitica from pig tonsils. We tested a panel of 26 bacterial strains on YECA and compared it to PCA, CIN, and YeCM media. Detection of pathogenic Y. enterocolitica was carried out on 50 pig tonsils collected in one slaughter house. Enrichment was done in PSB and ITC broths. Streaking on YECA and CIN was done in direct, after 24H incubation of ITC, after 48H incubation of PSB and ITC. All the plates were incubated at 30°C during 24 hours. Presence of typical colonies on CIN and YECA was checked, and isolates were biotyped. Pathogenic Y. enterocolitica strains showed an important growth on YECA with small and red fuchsia colonies while biotype 1A exhibited very few violet colonies. Enrichment in ITC during 48H gave the best performance for detecting positive samples in pathogenic Y. enterocolitica, and YECA could detect directly pathogenic Y. enterocolitica strains (2, 3, and 4). Use of YECA in combination with ITC generates a time-saver by giving a positive test in 72H.

Evaluation of isolation methods for pathogenic Yersinia enterocolitica from pig intestinal content

The aim of this study was to evaluate the efficiency of four isolation methods for the detection of pathogenic Yersinia enterocolitica from pig intestinal content. The four methods comprised of 15 isolation steps using selective enrichments (irgasan-ticarcillin-potassium chlorate and modified Rappaport broth) and mildly selective enrichments at 4 or 25 degrees C. Salmonella-Shigella-desoxycholate-calcium chloride agar, cefsulodin-irgasan-novobiocin agar were used as plating media. The most sensitive method detected 78% (53/68) of the positive samples. Individual isolation steps using cold enrichment as the only enrichment or as a pre-enrichment step with further selective enrichment showed the highest sensitivities (55-66%). All isolation methods resulted in high numbers of suspected colonies not confirmed as pathogenic Y. enterocolitica. Cold enrichment should be used in the detection of pathogenic Y. enterocolitica from pig intestinal contents. In addition, more than one parallel isolation step is needed. The study shows that depending on the isolation method used for Y. enterocolitica, the detected prevalence of Y. enterocolitica in pig intestinal contents varies greatly. More selective and sensitive isolation methods need to be developed for pathogenic Y. enterocolitica.

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.

Evaluation of a Combined Culture and PCR Method (NMKL-163A) for Detection of Presumptive Pathogenic Yersinia enterocolitica in Pork Products

A combined culture and PCR method for detection of pathogenic Yersinia enterocolitica in food (NMKL-163A) was evaluated by testing samples of artificially and naturally contaminated pork. The performance of the pre-PCR sample treatment, buoyant density centrifugation, was first compared with two commercially available methods (DNeasy tissue kit and PrepMan). We found that similar sensitivity was reached (i.e., 25 CFU/g of food was detected by single PCR) with the buoyant density centrifugation and the DNeasy Tissue kit when tested on overnight enrichments. However, the DNeasy tissue kit was superior when tested on nonenriched homogenates; the detection limit was 25 CFU/g in minced beef by single PCR and 25 CFU/g in sausage by nested PCR. We then analyzed 100 raw minced pork samples. Thirty-five tested positive for presumptive pathogenic Y. enterocolitica when analyzed by the NMKL-163A method, whereas none tested positive when analyzed in parallel by a standard culture method (ISO 10273). We also analyzed 97 samples of cold-smoked pork sausage, of which approximately 11% tested positive by the NMKL-163A method. This study showed that sensitivities such as those obtained by nested PCR were required for detection of the pathogen in naturally contaminated samples, and therefore the nested PCR primers, which are included in the NMKL-163A method only as an option, need to be validated and applied routinely.

A comparison of sample preparation methods for PCR detection of pathogenic Yersinia enterocolitica from ground pork using swabbing and slurry homogenate techniques

Two sample preparation methods for multiplex polymerase chain reaction (PCR) for detection of plasmid-bearing virulent Yersinia enterocolitica (YEP +) from ground pork were compared. Two sets of ground pork samples were inoculated with 10, 1, and 0.5 CFU/cm 2 of a YEP + strain, one set was swabbed and the second set was dispersed into a slurry homogenate. Both swab and slurry homogenate samples were enriched in sterile Whirl Pak bags containing modified trypticase soy broth for 48 h at 12 °C. From the enriched swab samples, the bacterial cells were pelleted, washed, boiled in sterile distilled water, and treated with proteinase K to prepare cell lysates to use as a DNA template. Since slurry homogenate samples contained food material, DNA extraction was performed using a commercial kit. The DNA from cell lysates and from extracted slurry homogenate samples were evaluated as templates for multiplex PCR employing primers for the chromosomal ail and plasmid virF genes. The enrichment of the YEP + strain was more efficient using the sponge-swabbed samples than the slurry homogenate samples at all three inoculum levels tested. It was necessary to dilute the DNA extracted from slurry homogenate to determine the optimal concentration of each sample for PCR amplification. No amplification signal was detected using undiluted DNA, possibly due to DNA inhibitors present in the slurry homogenate that were not removed in the process of extraction. However, DNA could be detected in undiluted cell lysates from swab samples. Thus, the cell lysates from swab samples are more advantageous than DNA extracted from ground pork slurry homogenate samples for the PCR assay.

Multiplex-PCR에 의한 먹는샘물 및 야채류로부터의 병원성 Yersinia enterocolitica의 신속검출

본 연구는 식품에 존재하는 Y. enterocolitica균의 신속한 점출방법을 조사하기 위하여 이균에 특이적인 ail, yst 및 virF 유전자와 Yersinia 속균을 구별하는 subgenus specific Y16S primer를 도입하여 multiplex PCR을 수행하였으며 Y. enterocolitica균의 검출 민감도와 특이도 및 먹는 샘물과 야채류에서의 적용실험을 각각 조사하였다. PCR특이성 실험에서는 Y. enterocolitica ATCC 27729균은 356 bp(ail), 134 bp(yst) 및 200 bp(Y16S) 3종의 유전자에 대한 DNA 증폭밴드를 나타내었으며, Y. entercolitica ATCC 9610 및 ATCC 23715는 yst와 Y16S 2종에만 증폭을 보였다. 반면에 기타 비병원성 Yersinia균인 Y. frederikseni, Y. intermedia, Y. kristenseni, Y. pseudotuberculosis 등은 Y16S에만 DNA 밴드를 나타내었으나 기타 세균인 E. coli ATCC 25392, Shi, dysenteri,m S. aureus ATCC 25923, L. monocytogenes ATCC 19111균에서는 어떤 primer에서도 특이 DNA 밴드를 확인할 수 없었다. PCR 민감도는 다른 유전자에 비하여 yst 유전자가 Y. enterocolitica균에 가장 높은 민감도를 나타내었고, Y16S 유전자는 속과 종에 관계없이 높은 민감도를 나타내었다. 따라서, 먹샘물이나 야채류에 대한 병원성 Yersinia균의 지표유전자로서 속을 대표하는 Y16S유전자와 종을 대표하는 yst유전자를 선정하였다. 수질 중 Y. enterocolitica의 검출한계를 알아보기 위하여 일반세균수가 3600 CFU/mL정도 함유된 먹는 샘물의 경우, DNA를 분리하지 않고 단순 열처리한 배양액을 DNA주형으로 사용하여 multiplex-PCR을 실시한 결과, Y16S와 yst 유전자 모두 7×10¹ CFU/mL 수준까지 검출할 수 있었으며, 일반세균수가 2.5×105CFU/g 정도 함유된 상치는 7 및 7×10¹ CFU/g, 일반세균이 7.1×10⁴ CFU/g정도 함유된 양송이버섯은 7 및 7×10¹ CFU/g 수준까지 검출할 수 있었다. 본 연구에서 나타난 바와 같이, 수질이나 야채류에서 Y16S와 yst 유전자를 혼합하여 Y. enterocolitica를 검출할 경우, DNA를 분리하지 않고 직접 whole cell을 lysate하여 DNA주형으로 사용하여도 2차 PCR을 수행할 경우에는 증균과정을 하지 않고도 민감도를 증진시키면서 신속하게 효율적으로 원하는 대상균을 검출할 수 있는 것으로 나타났다.

Detection of pathogenic Yersinia enterocolitica by a swab enrichment PCR procedure.

Detection of pathogenic Yersinia enterocolitica by a swab enrichment PCR procedure.

A Method Combining Immunomagnetic Separation Followed by Planting and Polymerase Chain Reaction Assay for the Detection of Pathogenic Yersinia enterocolitica from Food and Fecal Samples

A Method Combining Immunomagnetic Separation Followed by Planting and Polymerase Chain Reaction Assay for the Detection of Pathogenic Yersinia enterocolitica from Food and Fecal Samples

A combined culture and PCR method for detection of pathogenic Yersinia enterocolitica in food

A combined method based on traditional culturing, buoyant density centrifugation, (BDC), and polymerase chain reaction (PCR) techniques for detection and identification of pathogenic Y. enterocolitica in food was developed and evaluated. An internal control, which was added in each PCR-tube and co-amplified by the same primer pair as the pathogen, monitored false-negative PCR results. The sample preparation step, BDC, was used to remove PCR inhibiting food substances and to concentrate the Y. enterocolitica cells. Single PCR with a chromosomal gene ( ail) as target was chosen for screening the samples. The method was tested on naturally and artificially contaminated food samples. In three different food samples, processed meat (brawn), unprocessed beef and minced pork, inoculated with 10 cfu pathogenic Y. enterocolitica per gram, Y. enterocolitica was detected and cultural bacteria indicated within 18 h of enrichment.

Detection of pathogenic Yersinia enterocolitica in enrichment media and pork by a multiplex PCR: a study of sample preparation and PCR-inhibitory components

A multiplex PCR assay including sample preparation was developed to detect viable pathogenic strains of Yersinia enterocolitica in PCR-inhibitory samples, such as pork and enrichment media. The method developed was used to simultaneously detect the plasmid-borne virulence gene yadA and a Yersinia-specific region of the 16S rRNA gene. According to an auto-agglutination test for virulence-plasmid-bearing strains of Y. enterocolitica, all potential pathogenic strains tested were detected by the assay. A DNA extraction procedure, an aqueous two-phase system composed of polyethylene glycol 4000 and dextran 40 and a buoyant density centrifugation method, based on Percoll, were compared with regard to their efficiency in separating Yersinia enterocolitica from PCR inhibitors originating from enrichment media and pork. Using the density gradient centrifugation method resulted in a detection level of 4.0·10 2 CFU Y. enterocolitica per ml enrichment media. To ensure detection of viable bacteria a short enrichment step was included in the sample preparation together with the density gradient centrifugation. When this sample treatment method was evaluated with a selective enrichment medium together with a background flora inoculated with approximately 1.0·10 1 CFU per ml of Y. enterocolitica and incubated at 25°C, a positive PCR result was obtained after 6 to 8 h. Our results indicate that selective enrichment followed by buoyant density gradient centrifugation provides a convenient and user-friendly sample preparation method prior to PCR.

In situ localisation of Yersinia enterocolitica by catalysed reported deposition signal amplification.

AIM: The sensitive detection of pathogenic Yersinia enterocolitica in paraffin embedded tissue sections by in situ hybridisation (ISH). METHODS: Y enterocolitica infected cell lines, rat spleens, and patient biopsy specimens...

Detection of pathogenic Yersinia enterocolitica in milk and pork using a DIG-labelled probe targeted against the yst gene

A PCR-amplified, digoxigenin (DIG)-labelled yst probe was evaluated in naturally and artificially contaminated pork and milk samples for its ability to detect and differentiate between pathogenic and nonpathogenic strains of Yersinia enterocolitica following enrichment in irgasan-ticarcillin-chlorate (ITC) enrichment broth. A total of 44 raw pork samples were obtained from one meat-processing factory and two local retail stores. Y. enterocolitica serogroup O:3/biovar 4 was isolated from 6 of the 44 (14%) pork samples tested. The hybridization results were in good agreement when compared with those from standard biochemical and serological tests. The lowest concentration detectable by a colony hybridization technique in the milk samples inoculated with Y. enterocolitica O:8 PA-A corresponded to 10 cfu/ml in the original sample. No pathogenic serovars were detected in 6 raw milk and 3 pasteurized milks tested. The results of this investigation demonstrate that the DIG-labelled yst probe is a reliable tool for rapid detection of pathogenic Y. enterocolitica in naturally and artificially contaminated food samples.

Detection and occurrence of waterborne bacterial and viral pathogens

Detection and occurrence of waterborne bacterial and viral pathogens

Specific detection of pathogenic Yersinia enterocolitica by two-step PCR using hot-start and DMSO

A pair of polymerase chain reaction (PCR) primers, YC1 and YC2, selected from the sequence of the invasin locus (inv) of Y. enterocolitica, has been evaluated for specific detection of pathogenic Y. enterocolitica by PCR. The primers were hybridized at high stringency conditions to DNA from 65 pathogenic Y. enterocolitica, 16 non-pathogenic Y. enterocolitica, 18 other Yersinia and 124 non-Yersinia strains. YC2 hybridized to the pathogenic Y. enterocolitica only, while YC1 hybridized weakly to nine non-Yersinia as well.In a PCR with annealing at 64°C all Y. enterocolitica, pathogenic and non-pathogenic, were positive. However, DNA from 60 non-Y. enterocolitica was amplified. With annealing at 72°C, 10 non-pathogenic Y. enterocolitica and 41 non-Y. enterocolitica were positive. When a two-step PCR assay with annealing at 72°C, hot-start and 1% dimethylsulphoxide (DMSO) were used, only DNA from the pathogenic Y. enterocolitica were amplified. The limit of detection was shown for four different strains to be less than 10 cells per PCR tube.

Detection of pathogenic Yersinia enterocolitica in foods and water by immunomagnetic separation, nested polymerase chain reactions, and colorimetric detection of amplified DNA

A two-step polymerase chain reaction (PCR) procedure with two nested pairs of primers specific for the yadA gene of Yersinia enterocolitica was developed. The PCR assay identified all common pathogenic serogroups (O:3, O:5,27, O:8, O:9, O:13, and O:21) from three continents and differentiated pathogenic Y. enterocolitica from Y. pseudotuberculosis and from a variety of nonpathogenic yersiniae representing 25 serogroups and four species. The performance of the method was evaluated with seeded food and water samples. We compared two procedures for sample preparation prior to PCR: one was based on immunomagnetic separation of the target bacteria from the sample, using magnetic particles coated with immunoglobulin antibodies to Y. enterocolitica serogroup O:3, and the other method consisted of a series of centrifugation steps combined with proteinase treatment. Regardless of the method used, the PCR assay was capable of detecting 10 to 30 CFU/g of meat in 10(6)-fold excess of indigenous bacteria. When the samples were enriched overnight in a nonselective medium, the sensitivity was increased to approximately 2 CFU/g, except for samples with an extremely high background flora (> 10(7) CFU/g). We compared gel electrophoretic detection of PCR products with a colorimetric detection method designated DIANA (detection of immobilized amplified nucleic acids), which enabled easy visualization of amplified fragments in a microtiter plate format with an optical density reader. DIANA and gel electrophoresis showed complete concordance in their discrimination between positive and negative samples. The combination of immunomagnetic separation, nested PCR, and DIANA makes possible the development of a fully automated analytic process which requires a minimum of laboratory manipulations.