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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
State Key Laboratory of Applied Microbiology, South China (the Ministry–Province Joint Development), Provincial Key Laboratory of Microbiology Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou, 510070, China
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.
Yersinia enterocolitica is a common foodborne pathogen causing acute gastroenteritis, enterocolitis, and mesenteric lymphadenitis as well as extraintestinal disorders in humans (
EFSA-ECDC (European Food Safety Authority and European Center for Disease Prevention and Control). The European Union summary report on trends and sources of zoonoses, zoonotic agents and food borne outbreaks in 2010..
). Nonpathogenic strains are frequently isolated from food samples, and it is therefore essential to distinguish truly pathogenic strains from apathogenic strains. The identification of pathogenic Y. enterocolitica is traditionally based on phenotypic tests such as calcium-dependent growth at 37°C, Congo red binding (
). Plasmid-borne virulence factors include the virF gene, which encodes transcriptional activators of Y. enterocolitica outer membrane protein (yop) regulation and yadA gene involved in autoagglutination, serum resistance, and adhesion (
). Therefore, rapid and accurate detection of pathogenic Y. enterocolitica should rely on virulent factors not only from the chromosome, but also from the plasmid.
Yersinia enterocolitica is able to survive and proliferate in frozen or refrigerated conditions and is widely distributed in milk samples (
). Frozen or refrigerated dairy products are popular with consumers. In China, 296 strains of Y. enterocolitica were collected from humans with diarrhea, pigs, rodents, sheep, and dogs between 1984 and 2011 for analysis of variation and changes in serotypes, biotypes, and genetic typing (
reported possible use of ail and foxA polymorphisms for detection of Y. enterocolitica strains isolated from humans with diarrhea, animals, and environments. However, information about prevalence of pathogenic Y. enterocolitica in frozen or refrigerated dairy products is not available.
The aim of this study was to develop a duplex PCR targeting the virF and ail genes for determination of prevalence of pathogenic Y. enterocolitica in refrigerated or frozen dairy products. In addition, we developed a dot hybridization assay based on virF or ail genes to further determine the specificity of the duplex PCR. Thereafter, the detection limits of the duplex PCR and dot hybridization were determined. Finally, virulent and avirulent Y. enterocolitica isolates were tested in commercial frozen or refrigerated dairy samples (n = 88) by the duplex PCR and the
China National Standard. 2008. GB/T.4789.8-2008: Microbiological examination of Food hygiene—Examination of Yersina enterocolitica. Chinese National Standardization Management Committee, Beijing, China. (in Chinese)
All strains used in this study were from Guangdong Provincial Key Laboratory of Microbiology Culture Collection and Application (Guangzhou, China). Strains included 72 Y. enterocolitica, including 2 pathogenic strains (CMCC 52204 and CY39), 22 Escherichia coli, 16 Salmonella, 1 Enterobacter cloacae, 16 Listeria monocytogenes, 9 Staphylococcus aureus, 10 Aeromonas hydrophila, and 6 Aeromonas veronii. Yersinia enterocolitica CY39 and the type strain CMCC 52204 were used as positive controls for detection of ail+/virF+ and ail+, respectively. The non-Yersinia enterocolitica strains were used as negative controls.
Extraction of Genomic and Plasmid DNA of Y. enterocolitica and Other Strains
All species and strains listed above were inoculated into brain heart infusion broth (BHI, Huankai, Guangzhou, China) at 28°C for 36 h (for Y. enterocolitica) or 37°C for 24 h (for other strains), respectively. One milliliter of enrichment culture (about 6.5 × 109 cfu/mL for Y. enterocolitica, and 3.5 to 7.8×109 cfu/mL for other species) was used for extraction of genomic and plasmid DNA using the nucleic acid purification kit (DSBIO, Guangzhou, China) and high plasmid extraction kit (DSBIO), respectively, according to the manufacturer’s instructions. The DNA templates were dissolved in Tris-EDTA buffer (30 µL) and stored at −30°C until further use.
Detection of Pathogenic Y. enterocolitica by Duplex PCR
. For the duplex PCR, 2 pairs of primers were used: (forward): 5′-taatgtgtacgctgcgag-3′, (reverse): 5′-gacgtcttacttgcactg-3′ for ail amplification; and (forward): 5′-ggcagaacagcagtcagacata-3′, (reverse): 5′-ggtgagcatagagaatacgtcg-3′ for virF amplification. The PCR mixture (25 µL) consisted of 1.0 µL of primers (10 µM) for each, 200 µM deoxynucleotide triphosphates, 2 U of Taq DNA polymerase, 2.5 µL of 10× buffer, 3 µL of DNA template, and double-distilled water. The duplex PCR was performed as follows: denatured for 2.0 min at 95°C, followed by 35 cycles of 1.0 min at 94°C, 45 s at 58°C, and 1.0 min at 72°C; and final extension at 72°C for 7.0 min. The amplicons were detected by 1.5% agarose gel electrophoresis with Goldview staining (0.008%, vol/vol, Beijing Solarbio Science & Technology Co. Ltd., Beijing, China), and were photographed under a UV transilluminator.
Labeling and Detection of Pathogenic Y. enterocolitica by Dot Hybridization
The virF (561 bp) and ail (351 bp) sequences from the positive strains were labeled using the DIG High Prime DNA Labeling and Detection Starter Kit I (Roche, Basel, Switzerland) according to the manufacturer’s instructions. The detection of dot hybridization was performed using Detection Starter Kit I (Roche) according to the manufacturer’s instructions. In brief, 1.0 µL of tested DNA template was spotted onto the nylon membrane, the membrane was placed onto 3-MM paper (Whatman PLC, Little Chalfont, UK) soaked with 2× saline sodium citrate, and the template DNA on wet membrane was fixed by crosslinking with UV light. After crosslinking, the membrane was rinsed briefly in double-distilled water and was allow to air dry. Thereafter, the membrane was transferred into a plastic container with DIG Easy Hyb (10 mL/100-cm2 filter; Roche) for prehybridization with gentle agitation for 30 min. Prehybridization solution was poured off and mixture of denatured labeled probe and preheated DIG Easy Hyb (3.5 mL/100-cm2 filter) was transferred onto the membrane for incubation for 4 h. After hybridization and stringency washes, the membrane was briefly washed (5 min) in washing buffer, and then incubated in 10 mL of blocking solution for 30 min and 10 mL of antibody solution for 30 min, washed with 10 mL of washing buffer twice for 15 min each time, and then equilibrated in 10 mL of detection buffer for 2 to 5 min. Finally, the membrane was incubated in 2 mL of freshly prepared color nitro-blue tetrazolium chloride/ 5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt (NBT/BCIP) substrate solution in a hybrid tube in the dark. When desired spot or band intensities were achieved, the membrane was washed for 5 min with 50 mL of Tris-EDTA buffer to stop the reaction, and then the membrane was documented by photography.
Detection Limit for Artificially Contaminated Dairy Samples
Refrigerated yogurt (1.0 g) negative for Y. enterocolitica by the
China National Standard. 2008. GB/T.4789.8-2008: Microbiological examination of Food hygiene—Examination of Yersina enterocolitica. Chinese National Standardization Management Committee, Beijing, China. (in Chinese)
) method was mixed with 8 mL of sterile distilled water in a tube. Serial 10-fold dilutions from 6.5 × 109 to 6.5 × 100 cfu/mL were made, and 1.0 mL of each dilution was added to 9.0 mL of reconstituted yogurt. One milliliter of artificially contaminated yogurt was subject to genomic and plasmid DNA extraction using the same kits as used extraction of DNA from bacterial species. Genomic and plasmid DNA were stored at −30°C pending analysis. The PCR amplification and dot hybridization protocol were performed as described above.
Detection Limit of PCR Combined with Nonselective Enrichment
Brain heart infusion broth (5 mL) was inoculated with 4.5 to 5.7 cfu of Y. enterocolitica CY39 and incubated at 28°C with shaking (180 rpm) for 2, 4, 6, 8, 10, 12, 14, 16, and 18 h. The extraction of plasmid and genomic DNA from the different time-enrichment cultures (1.0 mL) and PCR amplification were performed as described above.
Detection of Y. enterocolitica in Commercial Frozen or Refrigerated Dairy
Eighty-eight frozen or refrigerated dairy samples including ice cream (n = 20), refrigerated yogurt (n = 14), milk popsicles (n = 18), refrigerated raw milk (n = 14), and frozen cheese (n = 22) were collected from supermarkets or retail shops. The samples (25 g) were incubated into 225 mL of modified phosphate saline buffer and incubated at 28°C for 48 h. Then, 1 mL of culture was subjected to extraction of template DNA (genomic DNA and plasmid DNA). Yersinia enterocolitica and pathogenic Y. enterocolitica were isolated and detected by the
China National Standard. 2008. GB/T.4789.8-2008: Microbiological examination of Food hygiene—Examination of Yersina enterocolitica. Chinese National Standardization Management Committee, Beijing, China. (in Chinese)
Phenotyping of Pathogenic Y. enterocolitica Strains
Yersinia enterocolitica phenotypes were grouped by biotyping (lipase, esculin, salicin, indole, xylose, and trehalose tests) and serotyped, using commercial serum, as O:3, O:5, O:8, O:9, and O:1, 2 (Denka Seiken, Tokyo, Japan) according to the manufacturer’s instructions.
Results and Discussion
Yersinia enterocolitica is a common foodborne pathogen causing human cases of yersiniosis through consumption of contaminated food (
). Consequently, detection of pathogenic Y. enterocolitica in frozen dairy samples is important in assuring microbial safety.
In this study, a duplex PCR targeting plasmid-borne virF and chromosome-borne ail genes was developed to simultaneously detect virulent Y. enterocolitica strains in frozen or refrigerated dairy samples. The expected fragments (561 bp for virF, 351 bp for ail) were successfully amplified from 12 out of 72 Y. enterocolitica isolates, but not in any non-Y. enterocolitica strains by our new duplex PCR. In Figure 1, among 12 pathogenic Y. enterocolitica, 6 pathogenic Y. enterocolitica strains were positive for both ail and virF genes, and 6 strains were positive for the ail gene only. As seen in Figure 2, detection by dot hybridization was consistent with results detected by the duplex PCR, which further confirmed the specificity of the duplex PCR method for detection of virulent Y. enterocolitica. Using the dot hybridization assay, we achieved a detection limit of 6.5 × 103 cfu/mL (Figure 3A). A detection limit of 1,000 cfu by microarray hybridization targeting virulence genes in pasteurized whole milk was described by
. As seen in Figure 3B, our duplex PCR could directly detect 6.5 × 102 cfu/mL of Y. enterocolitica in artificially contaminated dairy samples. A detection level of 4.0 × 102 cfu of Y. enterocolitica per mL of enrichment medium was obtained by a duplex PCR targeting yadA and 16S rRNA genes in combination with the density gradient centrifugation method (
Detection of pathogenic Yersinia enterocolitica in enrichment media and pork by a multiplex PCR: A study of sample preparation and PCR-inhibitory components.
) reported that as few as 102 cfu could be successfully detected in ground pork in the presence of 105 to 106 cfu of other species by a single PCR targeting ail or virF. However, in that study, only 16 of 25 pathogenic Y. enterocolitica strains simultaneously yielded positive results for both genes. To determine the influence of enrichment on the detection limit (Figure 4), the expected amplicons were amplified by the duplex PCR from the artificially dairy sample inoculated with 4.5 to 5.7 cfu of Y. enterocolitica /5 mL of brain heart infusion broth after 6 h of nonselective enrichment at 28°C. In an earlier study using selective enrichment at 25°C, a positive PCR result was obtained from selective enrichment medium with a background flora inoculated with approximately 1.0 × 101 cfu/mL Y. enterocolitica after 6 to 8 h at 25°C (
Detection of pathogenic Yersinia enterocolitica in enrichment media and pork by a multiplex PCR: A study of sample preparation and PCR-inhibitory components.
). The current study also indicated that the duplex PCR was specific and sensitive for detection of pathogenic Y. enterocolitica when combined with an enrichment step.
Figure 1Detection of pathogenic Yersinia enterocolitica strains using duplex PCR. M = marker; lanes 1 to 6 = pathogenic Y. enterocolitica strains containing both ail and virF genes; lanes 1′ to 6′ = pathogenic Y. enterocolitica strains containing ail gene only; P = positive control, N = negative control.
Figure 2Detection of pathogenic Yersinia enterocolitica strains by dot hybridization assay on ail and virF genes. A-ail = 6 pathogenic Y. enterocolitica strains containing ail gene only; B-ail and B-virF = 6 pathogenic Y. enterocolitica strains containing both ail and virF virulence genes; P = positive control, N = negative control. Dots 1 to 6 correspond to the 6 pathogenic Y. enterocolitica strains in each set.
Figure 4Detection of duplex PCR for ail and virF genes combined with nonselective enrichment. M = marker; N = negative control; lanes 1 to 8 = enrichment for 18, 16, 14, 12, 10, 8, 6, and 4 h at 28°C, respectively.
Surveillance of Y. enterocolitica is important for risk analysis and assessment of frozen or refrigerated dairy products. In Pennsylvania, Y. enterocolitica was detected in 248 (1.2%) milk samples (
). In the current study (Table 1), out of 88 dairy samples, pathogenic Y. enterocolitica was detected in 5 (5.7%) samples, of which milk popsicles had the highest prevalence (2/18; 11.1%), followed by refrigerated raw milk (1/14; 7.1%), ice cream (1/20; 5%), frozen cheese (1/22; 4.5%), and refrigerated yogurt (0/14; 0.0%), compared with 16.7, 15, 14.3, 14.3, and 13.6% of milk popsicles, ice cream, refrigerated yogurt, refrigerated raw milk, and frozen cheese samples shown to be positive for Y. enterocolitica by the
China National Standard. 2008. GB/T.4789.8-2008: Microbiological examination of Food hygiene—Examination of Yersina enterocolitica. Chinese National Standardization Management Committee, Beijing, China. (in Chinese)
) reported that Y. enterocolitica from biotypes 1B, 2, 3, 4, and 5 were pathogenic in humans, which was consistent with our results. The following O serotypes were detected among these pathogenic Y. enterocolitica strains: O:8 (7 isolates), O:5 (4 isolates), O:3 (3 isolates), and O:9 (2 isolates), but 1 isolate was negative for the O serotype (O:3, O:5, O:8, O:9, and O:1, 2). Serotype groups associated with human infections (O:3, O:5, O:8, and O:9) were previously described by
China National Standard. 2008. GB/T.4789.8-2008: Microbiological examination of Food hygiene—Examination of Yersina enterocolitica. Chinese National Standardization Management Committee, Beijing, China. (in Chinese)
The survey in commercial dairy samples indicated that contamination of Y. enterocolitica is relatively severe and should be emphasized. In addition, only 1 of 5 pathogenic Y. enterocolitica strains bore both the ail and virF genes. Previous studies have reported that all Y. enterocolitica strains harboring the ail gene always carry the yst gene (
). In present study, we found that the plasmid-borne virF did not occur alone in pathogenic Y. enterocolitica and was always accompanied by the ail gene. The developed duplex PCR was able to differentiate pathogenic Y. enterocolitica from apathogenic strains regardless of biotype or serotype. The serotypes O:3, O:5, O:8, and O:9 and biotypes 1B, 2, 3, 4, and 5 were considered pathogenic (
). The method described here could also distinguish pathogenic Y. enterocolitica strains with or without the virulence plasmid harboring the virF gene. Furthermore, instability of the virulence plasmid has been reported previously (
) and the plasmid can be easily lost under conditions of alkaline pH (e.g., pH 9.0; data not shown). Consequently, plasmid-borne virulence genes are not appropriate as the sole marker for specifically detecting pathogenic Y. enterocolitica strains in food samples.
Conclusions
In this study, we developed a duplex PCR targeting ail and virF virulence genes that could specifically and sensitively detect pathogenic Y. enterocolitica in frozen or refrigerated dairy products. A dot hybridization assay further confirmed the specificity of the duplex PCR. When combined with an enrichment step, the duplex PCR had high sensitivity. Detection of pathogenic Y. enterocolitica in commercial frozen or refrigerated dairy samples indicated that the duplex PCR was reliable for detection of pathogenic Y. enterocolitica and for routine inspection and monitoring in refrigerated or frozen dairy samples.
Acknowledgments
We gratefully acknowledge the financial support of the National Natural Science Foundation of China (Beijing, China; 31201292) and Guangdong Province, Chinese Academy of Science comprehensive strategic cooperation project (Guangzhou, China; 2012B090400017).
References
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Direct detection and isolation of plasmid-bearing virulent serotypes of Yersinia enterocolitica from various foods.
China National Standard. 2008. GB/T.4789.8-2008: Microbiological examination of Food hygiene—Examination of Yersina enterocolitica. Chinese National Standardization Management Committee, Beijing, China. (in Chinese)
Detection of pathogenic Yersinia enterocolitica in enrichment media and pork by a multiplex PCR: A study of sample preparation and PCR-inhibitory components.
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