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A Highly Specific One-step PCR Assay for the Rapid Discrimination of Enteropathogenic Yersinia enterocolitica from Pathogenic Yersinia pseudotuberculosis and Yersinia pestis
System. Appl. Microbiol. 24, 285–289 (2001) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/sam
A Highly Specific One-step PCR – Assay for the Rapid Discrimination of Enteropathogenic Yersinia enterocolitica from Pathogenic Yersinia pseudotuberculosis and Yersinia pestis THORSTEN ARNOLD1, ANDREAS HENSEL1, RALF HAGEN2, STOJANCA ALEKSIC3, HEINRICH NEUBAUER2 and HOLGER C. SCHOLZ1 1
Institute of Animal Hygiene and Public Veterinary Health, Leipzig, Germany Institut für Mikrobiologie, Sanitätsakademie der Bundeswehr, München, Germany 3 Institute for Hygiene, National Reference Center for Yersiniosis, Hamburg, Germany 2
Received May 25, 2001
Summary Based on differences within the yopT-coding region of Yersinia. enterocolitica, Y. pseudotuberculosis and Y. pestis, a rapid and sensitive one-step polymerase chain reaction assay with high specificity for pathogenic Y. enterocolitica was developed. By this method pathogenic isolates of Y. enterocolitica can be easily identified and discriminated from other members of this genus. The entire coding sequence of the yopT effector gene of Y. pseudotuberculosis Y36 was determined. Key words: Yersinia – yopT – PCR – diagnostic
Introduction Yersinia enterocolitica is a common enteric pathogen responsible for a variety of disorders in humans and animals (NEUBAUER et al., 2001a; NEUBAUER et al., 2001b). Animals are often asymptomatically infected, but do represent a potential source for human infection (FREDRIKSSON-AHOMAA et al., 1999; RAMIREZ et al., 2000; BORCH et al., 1996). When transmitted to humans, pathogenic strains of Y. enterocolitica can cause an acute gastroenteritis, enterocolitis, and mesenteric adenitis, as well as a variety of extra intestinal disorders (TAUXE et al., 1987; HOOGKAMP-KORSTANJE and KONING, 1990; ABDEL-HAQ et al., 2000). Among the six biotypes, and more than 28 serotypes of Y. enterocolitica described to date, only few clones are considered to be pathogenic. Virulence is strictly associated with the presence of the (67–72 kb) Yersinia virulence plasmid (CORNELIS et al., 1998) although non-plasmid carrying Yersinia isolates have been isolated from clinical samples (GRANT et al., 1998). Phenotypic differentiation between pathogenic and non-pathogenic strains of Y. enterocolitica and other Yersinia strains necessitates a complex combination of biochemical and serological tests which have been reported to
provide inconsistent results and to be time consuming (NEUBAUER et al., 2001c; WAAGE et al., 1999; VISHNUBHATLA et al., 2001). Markers for pathogenicity have been attributed to both, the chromosome and the Yersinia virulence plasmid (KWAGA et al., 1992; IBRAHIM et al., 1997; NILSSON et al., 1998). Consequently, various different DNA probes and PCR assays were developed to detect the organism in environmental samples and to differentiate between virulent and avirulent strains. However, so far, both types of markers (chromosomal and plasmid borne) must be used in combination to distinguish pathogenic from non-pathogenic Yersiniae, and Y. enterocolitica from Y. pseudotuberculosis (IBRAHIM et al., 1997; NAKAJIMA et al., 1992). In addition, problems occurred when multiple primer combinations were used in a single PCR reaction (WEYNANTS et al., 1996). Recently, a PCR assay based upon partial 16S rDNA amplification was developed to distinguish between Y. enterocolitica and Y. pseudotuberculosis (NEUBAUER et al., 2000). However, due to cross reactions within the genus Yersiniae, this method requires biochemical characterization of the isolate before PCR. 0723-2020/01/24/02-285 $ 15.00/0
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In this study we describe a specific, sensitive and rapid one-step PCR assay based on the plasmid-located virulence gene yopT to detect plasmid carrying Y. enterocolitica in pure cultures and artificially contaminated feces. The assay is also suitable to differentiate between pathogenic strains of Y. enterocolitica, Y. pseudotuberculosis and Y. pestis.
Material and methods Bacterial strains and growth conditions Y. enterocolitica Y11 (DSM 13030) was obtained from the Deutsche Sammlung von Mikroorganismen, Baunschweig, Germany. Y. pseudotuberculosis Y36, a virulent strain isolated from a guinea pig, was provided by Dr. G. Wolf, LMU Munich. For non-selective cultivation cells were grown in Luria Bertiani (LB) medium. For selective enrichment of Y. enterocolitica from feces, cells were grown in Yersinia selective medium (Oßmer). Inactivated Y. pestis EV76, Burkolderia mallei and B. pseudomallei, Brucella abortus and Francisella tularensis were provided by Dr. Niederwöhrmeier, WIS, Munster, Germany. Further inactivated Y. pestis strains were provided by Dr. Carniel, Institut Pasteur, Paris, France or J. Prior, CBD, Porton Down, UK. Bacteria of these 17 strains (108–109 bacteria/ml) were inactivated by heat before sample preparation. Cloning and sequencing of yopT from Y. pseudotuberculosis Y36 Y. pseudotuberculosis Y36 was grown at 37 °C on Luria Bertiani (LB) agar plates. The primer combination YopT-ff1 (5′ATGGACAGTATTCACGGACAC-3′) and YopT-rr1 (5′-TAAACCTCCTTGGAGTCAAATG-3′) derived from the known yopT coding sequence of Y. enterocolitica (ATCC 9610) was used to amplify the entire coding region of yopT. Single colonies were suspended in 500 µl double deionized water (ddH20). The mixture was heated for 15 min at 94 °C, and placed on ice. After centrifugation (13.000 × g, 5 min), 3 µl of the supernatant were used as template in the PCR assay. The reaction was carried out in a final volume of 50 µl, containing 5 µl 10× PCR-reaction-buffer (Roche, Mannheim, Germany), 5 µl 2mM dNTP mix (Prime Zyme), 10 pmol of each primer, 0.5 U Taq DNA polymerase (Roche, Mannheim), and 3 µl of template DNA. Optimization of cycling conditions resulted in initial denaturation for 5 min at 94 °C followed by 30 cycles each consisting of 80 sec. min denaturation at 94 °C, 40 sec annealing at 59 °C, and 80 sec. min extension at 72 °C, and a final extension step of 7 min. Amplification was performed in a Perkin-Elmer GeneAmp PCR System 2400 (PE, Weiterstadt, Germany). 5 µl of the PCR product was run on a 1%
agarose gel at a constant voltage of 140 V in TAE buffer (40 mM Tris [pH 7.6], 20 mM acetic acid, and 1 mM EDTA). The gel was stained with ethidium bromide and the PCR product was visualized under UV light. The resulting PCR product of 969 bp was directly cloned into the vector pXcmI, exhibiting an T-overhang suitable for direct cloning of amplified PCR products. Its nucleotide sequence was determined. DNA sequencing of the insert was carried out with a LI-COR DNA sequencer model 4000. EMBL nucleotide databank analysis confirmed the presence of yopT in Y. pseudotuberculosis. Generation of Yersinia enterocolitica specific oligonucleotides and partial amplification of yopT fromY. enterocolitica A multiple alignment of the amplified sequence with the yopT coding sequence from Y. enterocolitica (derived from plasmid pYVe227), and Y. pestis (derived from plasmid pCD1) was carried out with ClustalW 1.8. Differences between the yopT coding region of the three species were used to design Y. enterocolitica specific oligonucleotides. The partial alignment of yopT used for the design of Y. enterocolitica specific oligonucleotides is shown in Fig 1. The primer pair yopT-fw1 (5′-TATGTGCACATTGGATTT3′) and yopT-r1 (5′-AATGATACATAGAATTTT-3′) was used to amplify a 478 bp fragment of yopT. Template preparation and detection of the PCR was carried out as described above. Optimization of cycling conditions resulted in initial denaturation for 5 min at 94 °C followed by 35 cycles each consisting of 1 min denaturation at 94 °C, 40 sec annealing at 43 °C, and 1 min extension at 72 °C, and a final extension step of 7 min. Detection of Y. enterocolitica in artificially contaminated feces Three grams of feces were mixed with 3 ml of bacterial suspensions (culture in mid-exponential growth phase) of Y. enterocolitica Y11 (DSM 13030) containing 3 × 109 to 3 × 101 cells and mixed by stirring for 5 min to obtain final concentrations of 109 to 101 bacteria per gram feces. The volume of each sample was adjusted to 10 ml with ringer-solution. 1 ml of this mixture was used for enrichment in either 9 ml of selective (Oßmer) or non-selective (Luria Bertiani) medium. Immediately after inoculation and after 2, 4, 6, and 24 hours of enrichment, 1ml of the suspension was centrifuged at 300 × g in an Eppendorf tube to remove crude particles. The supernatant was centrifuged again at 12.000 × g. The pellet was washed twice in sterile phosphate buffered saline and resolved in a final volume of 200 ml. 10 ml were directly used for the PCR reaction. For direct bacterial DNA-extraction from contaminated feces without foregoing enrichment, the QIAamp DNA Stool Mini Kit (Qiagen) was used.
Fig 1. Alignment of the yopT nucleotide sequence (coding strand) used for the construction of Y. enterocolitica specific oligonucleotides. The oligonucleotides for the specific amplification of yopT in Y. enterocolitica are shown in bold letters. The corresponding sequences of Y. pestis and Y. pseudotuberculosis are given in the alignment below.
PCR based identification and differentiation of Yersinia spp.
Results and Discussion YopT encodes a 35.5 kDa effector protein (YopT) which induces a cytotoxic effect in HeLa cells and macrophages. The effect on HeLa cells consists of disruption of the actin filaments and alteration of the cell cytoskeleton (CORNELIS et al., 1998; IRIARTE and CORNELIS, 1998). In previous studies yopT was detected on the plasmid of pathogenic Y. enterocolitica as well as in the agent of plague, Y. pestis, but was either not detected or reported to be absent from Y. pseudotuberculosis (CORNELIS et al., 1998; HEESEMAN and HENSEL, 2000). In this study the entire yopT coding sequence was proved to be present in Y. pseudotuberculosis Y36 and its sequence was determined. Computer assisted analysis of the sequencing data confirmed the presence of the yopT coding sequence on the plasmid of Y. pseudotuberculosis Y36 (Acc. No., AJ304833). Sequence comparison with Y. enterocolitica and Y. pestis revealed a number of mismatches between the three species. This finding was used to develop a PCR-based assay to specifically detect- and to discriminate pathogenic strains of Y. enterocolitica from Y. pseudotuberculosis and Y. pestis. In order to prove the specificity of the PCR assay, various pathogenic and nonpathogenic strains of Y. enterocolitica and Y. pseudotuberculosis of different serovar/biovar combinations were investigated. The bacterial strains used in this study are listed in Tab. 1. As negative controls, various type strains of Yersinia spp., and members of other bacteria causing similar infections in humans or being possible sample contaminants were included. The yopT specific PCR product of 478 bp was only amplified from DNA of plasmid bearing Y. enterocolitica. No PCR product was obtained with either DNA of other Yersiniae (pathogenic Y. pseudotuberculosis and non pathogenic Yersiniae) or DNA obtained from other bacteria (Tab. 1). Therefore, this assay is highly specific for plasmid bearing Y. enterocolitica. To examine the detection limit for the PCR assay, a series of 10-fold dilutions of Y. enterocolitica-suspension beginning with 108 cells/ml in ddH2O was carried out. A PCR product was yielded still at a concentration of 104 cells when cells were directly used as template without a foregoing proteaseK treatment. To improve the detection limit, cells from each dilution were suspended in lysis buffer containing a final concentration of 0.025% Tween 20 [Sigma], and 0, 2 mg/ml proteinaseK [Merck], and incubated for 1 h at 56 °C. Using these optimized conditions 102 cells were detected. To determine the influence of fecal inhibitors porcine feces were artificially contaminated with different amounts of Y. enterocolitica. During the entire cell preparation procedure no heat treatment was used in order to keep the natural flora of microorganisms of the feces. No PCR product was yielded in the negative control with uninoculated feces demonstrating the specificity of yopTfw1 and yopt-r1. While no PCR product was amplified immediately after inoculation, 2 and 6 h of enrichment in non-selective medium were sufficient to detect Y. enterocolitica with a minimum of 108 and 105 cells/g feces, respectively. After 24 h of enrichment in LB-medium prior
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to PCR, detection of Yersiniae was achieved with 102 cells/g feces (data not shown). Interestingly, the detection limit in selective medium was 104 to 103 cells/g feces, indicating a strong negative effect of selective enrichment. Direct extraction of bacterial DNA from feces (without enrichment) with the QIAmp DNA stool kit resulted in a detection limit of 106 cells/g feces. Therefore, it can be suggested that inhibitory substances are not completely removed during the DNA preparation procedure. The exact bacterial cell number in feces of Yersiniashedding patients is unknown and varies in different stages of infection. However, the low detection limit of 102 cells/g feces may permit the rapid and specific detection of pathogenic Yersina enterocolitica in feces of infected patients within 28 h (24 h of enrichment). The assay is highly discriminating for pathogenic Y. enterocolitica in a single PCR reaction. Rapid detection and discrimination of enteropathogenic strains of Y. enterocolitica from other Yersinia strains as well as from pathogenic members of the genus Enterobacteriaceae is important for diagnostic purposes and for the hygiene control in food production. Most of the commercially available identification systems are time-consuming, and the sensitivity at species level is unsatisfying (WAAGE et al., 1999; LINDE et al., 1999; NEUBAUER et al., 1998; NEUBAUER et al., 2001b; VISHNUBHATLA et al., 2001). The detection of chromosomal virulence markers by PCR like ail, inv or yst (FENG et al., 1992; FENWICK and MURRAY, 1991; IBRAHIM et al., 1997; VISHNUBHATLA et al., 2000; JOURDAN et al., 2000) does not reflect the actual pathogenicity of an isolate. Although the mentioned and various other chromosomally located genes contribute to virulence, in most cases pathogenicity is associated with the presence of the virulence plasmid. Only few virulent isolates lacking known virulence genes (chromosomal and plasmid) have been described (GRANT et al., 1998). In this case the molecular mechanism of virulence is not known, but it can be assumed that the clonality of the isolates used was doubtful. Therefore, it can be postulated that markers specific for the virulence plasmid are most suitable to detect virulent strains. However, the diagnostic value of plasmid-encoded sequences as a diagnostic tool is discussed controversially as the plasmid can be lost during cultivation under laboratory conditions (FENWICK and MURRAY, 1998; IBRAHIM et al., 1992; WREN and TABAQCHALI, 1990). In our investigations, however, we observed that the plasmids of each strain tested were stable for at least 15 subcultivations (data not shown). In routine diagnosis when the pathogen is isolated from different specimens and identified without extensive subcultivation of the microorganism, the loss of the plasmid should not be observed. The sequences used to create Y. enterocolitca specific oligonucleotides might also be useful for the development of Y. pestis and Y. pseudotuberculosis specific PCR assays. In our investigations we could also show that the yopT based PCR assay is capable to specifically detect Y. enterocolitica in artificially contaminated feces in 24 hours with excellent correlation to that of routine bacterial diagnostics (data not shown). Future investigations
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Table 1. Listing of strains used in PCR analyses for partial yopT amplification. Species
serogroup/strain
source
No. of isolates tested
positive for yop T
pathogenic Yersinia strains (p+)1 (p+)2
O:3, O:5,27, O:9, O:2a.2b.3
H3, A4 H, A
21 19
+ –
Y. enterocolitica
(p–)
O:3, O:5 , O:6,30, O:13,7, O:4,33, O:7,8, O:41,43
H, A
92
–
Y. pseudotuberculosis Y. intermedia
(p–)
H, A
29 15
– –
1 1 1 1 1 1 1 1 1 1
– – – – – – – – – –
1 1 1 1 1 1 1 1 1 1
+ – – – – – – – – –
1 1 1 1 1 1 1 1 1 1 1 1 1 1
– – – – – – – – – – – – – –
Y. enterocolitica Y. pseudotuberculosis apathogenic Yersinia strains
O:3, O:5, O:16, O:17, O:40 , O:49,51
Yersinia pestis strains PKH 4519 Turquie 10-1 521 Senegal Thierno 523 Senegal Fay 524 Kenya 147 537 Congo Belge Lita PKR 25 564 Exu 21 567 Con.Bel.Elisab 678 Hamburg 9 695
(p+) (p– ) (p+) (p+) (p+) (p+) (p+) (p+) (p+) (p+)
Kurdistan Turkey Senegal Senegal Kenya Zaire Kurdistan Brazil Zaire Germany
Yersinia type strains Y. enterocolitica subsp. paleartica subsp. enterocolitica Y. pseudotuberculosis Y. intermedia Y. frederiksenii Y. kristensenii Y. aldovae Y. bercovieri Y. rhodei Y. mollaretti
(p+) (p– (p+)
DSM 213030 ATCC 9610 ATCC 29833 ATCC 29909 ATCC 33641 ATCC 33638 ATCC 35236 ATCC 43970 ATCC 43380 ATCC 43969
control group Salmonella enteritidis Salmonella typhimurium Salmonella typhimurium Salmonella senftenberg Enterococcus faecalis Enterobacter aerogenes Escherichia coli Escherichia coli Pseudomonas aeruginosa Staphylococcus aureus Brucella abortus Burkholderia pseudomallei Burkholderia mallei Francisella tularensis 1
p+ – virulence plasmid bearing p– – virulence plasmidless 3 H – human source 4 A – animal source 2
DT 104 SM 5569 775 W D7 / 63 DSM 30053 ATCC 11229 DSM 301 ATCC 15442 DSM 346 ATCC 15682 ATCC 15310
PCR based identification and differentiation of Yersinia spp.
will prove whether the developed assay is also suitable to rapidly detect pathogenic Y. enterocolitica in food or food products and in feces of infected patients. Based on our results we recommend this assay as a simple and time saving method to specifically identify virulence plasmid bearing Y. enterocolitica. Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (GRK-39/2) under project: “Schlachttierbelastung und Produktsicherheit”.
References 1. ABDEL-HAQ, N. M., ASMAR, B. I., ABUHAMMOUR, W. M., BROWN, W. J.: Yersinia enterocolitica infection in children Pediatr. Infect. Dis. J. 19, 954–8 (2000). 2. BORCH, E., NESBAKKEN, T., CHRISTENSEN, H.: Hazard identification in swine slaughter with respect to foodborne bacteria. Int. J. Food Microbiol. 30, 9–25 (1996). 3. CORNELIS, G. R., BOLAND, A., BOYD, A. P., GEUIJEN C., IRIARTE, M., NEYT, C., SORY, M. P., STAINIER I.: The virulence plasmid of Yersinia, an antihost genome. Microbiol. Mol. Biol. Rev. 62, 1315–1352 (1998). 4. FENG, P., KEASLER, S. P., HILL, W. E.: Direct identification of Yersinia enterocolitica in blood by polymerase chain reaction amplification. Transfusion 32, 850–854 (1992). 5. FENWICK, S. G., MURRAY, A.: Detection of pathogenic Yersinia enterocolitica by polymerase chain reaction [letter; comment]. Lancet 337, 496–497 (1991). 6. FREDRIKSSON-AHOMAA, M., HIELM, S., KORKEALA, H.: High prevalence of yadA-positive Yersinia enterocolitica in pig tongues and minced meat at the retail level in Finland. J. Food Prot. 62, 123–127. 1999. 7. GRANT, T., BENNETT-WOOD, V., ROBINS-BROWNE, R.M.: Identification of virulence-associated characteristics in clinical isolates of Yersinia enterocolitica lacking classical virulence markers. Infect. Immun. 66, 1113–20 (1998). 8. HEESEMANN, J., HENSEL, M.: Yersinia enterocolitica und Yersinia pseudotuberculosis, p. 216–218. In J. Hacker and J. Heesemann (eds.), Molekulare Infektionsbiologie. Spektrum Akademischer Verlag, Gustav Fischer, Heidelberg (2000). 9. HOOGKAMP-KORSTANJE, J.A., KONING, J.: Clinical aspects, diagnosis and therapy of Yersinia enterocolitica infections. Immun. Infekt. 18, 192–7 (1990). 10. IBRAHIM, A., LIESACK, W., GRIFFITHS M. W., ROBINSBROWNE, R. M.: Development of a highly specific assay for rapid identification of pathogenic strains of Yersinia enterocolitica based on PCR amplification of the Yersinia heatstable enterotoxin gene (yst). J. Clin. Microbiol. 35, 1636–1638 (1997). 11. IBRAHIM, A., LIESACK, W., STACKEBRANDT, E.: Polymerase chain reaction-gene probe detection system specific for pathogenic strains of Yersinia enterocolitica. J. Clin. Microbiol. 30, 1942–1947 (1992). 12. IRIARTE, M., CORNELIS, G. R.: YopT, a new Yersinia Yop effector protein, affects the cytoskeleton of host cells. Mol. Microbiol. 29, 915–929 (1998). 13. JOURDAN, A. D., JOHNSON, S. C., WESLEY, I. V.: Development of a fluorogenic 5’ nuclease PCR assay for detection of the ail gene of pathogenic Yersinia enterocolitica. Appl. Environ. Microbiol. 66, 3750–5 (2000). 14. KWAGA, J., IVERSEN, J. O., MISRA, V.: Detection of pathogenic Yersinia enterocolitica by polymerase chain reaction and digoxigenin-labeled polynucleotide probes. J. Clin. Microbiol. 30, 2668–2673 (1992).
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15. LINDE, H. J., NEUBAUER, H., MEYER, H., ALEKSIC, S.,LEHN N.: Identification of Yersinia species by the Vitek GNI card. J. Clin. Microbiol. 37, 211–214 (1999). 16. NAKAJIMA, H., INOUE, M., MORI, T., ITOH, K., ARAKAWA, E., WATANABE, H.: Detection and identification of Yersinia pseudotuberculosis and pathogenic Yersinia enterocolitica by an improved polymerase chain reaction method. J. Clin. Microbiol. 30, 2484-–2486 (1992). 17. NEUBAUER, H., SAUER, T., BECKER, H., ALEKSIC, S., MEYER, H.: Comparison of systems for identification and differentiation of species within the genus Yersinia. J. Clin. Microbiol. 36, 3366–3368 (1998). 18. NEUBAUER, H., SPRAGUE, L. D., SCHOLZ, H., HENSEL, A.: Yersinia enterocolitica infections in man. Berl. Münch. Tierärztl. Wschr. (BMTW) 114, 81–87 (2001a). 19. NEUBAUER, H., SPRAGUE, L. D., SCHOLZ, H., HENSEL, A.: Yersinia enterocolitica infections in animals. Berl. Münch. Tierärztl. Wschr. (BMTW) 114, 8–12 (2001b). 20. NEUBAUER, H., SPRAGUE, L .D., SCHOLZ, H., HENSEL, A.: The Diagnostics of Yersinia enterocolitica Infections: A Review on Classical Identification Techniques and New Molecular Methods. Berl. Münch. Tierärztl. Wschr. (BMTW) 114, 1–7 (2001c). 21. NEUBAUER, H., ALEKSIC, S., HENSEL, A., MEYER, H.: Identification of Yersinia enterocolitica within the genus Yersinia. Syst Appl Microbiol. 23, 58–62 (2000). 22. NILSSON, A., LAMBERTZ, S. T., STALHANDSKE, P., NORBERG, P., DANIELSSON-THAM, M. L.: Detection of Yersinia enterocolitica in food by PCR amplification. Lett. Appl. Microbiol. 26, 140–144 (1998). 23. RAMIREZ, E.I., VAZQUEZ-SALINAS, C., RODAS-SUAREZ, O. R., PEDROCHE, F. F.: Isolation of Yersinia from raw meat (pork and chicken) and precooked meat (porcine tongues and sausages) collected from commercial establishments in Mexico City. J. Food Prot. 63, 542–4 (2000). 24. TAUXE, R.V., VANDEPITTE, J., WAUTERS, G., MARTIN, S. M., GOOSSENS, V., DE MOL P., VAN NOYEN, R., THIERS, G.: Yersinia enterocolitica infections and pork: the missing link. Lancet 1, 1129–1132. 1987. 25. VISHNUBHATLA, A., FUNG, D. Y., OBERST, R. D., HAYS, M. P., NAGARAJA, T. G., FLOOD, S. J.: Rapid 5’ nuclease (TaqMan) assay for detection of virulent strains of Yersinia enterocolitica. Appl. Environ. Microbiol. 66, 4131–5 (2000). 26. VISHNUBHATLA, A., OBERST, R. D., FUNG, D. Y., WONGLUMSOM, W., HAYS, M. P., NAGARAJA, T. G.: Evaluation of a 5’nuclease (TaqMan) assay for the detection of virulent strains of Yersinia enterocolitica in raw meat and tofu samples. J. Food. Prot. 64, 355–60 (2001). 27. WAAGE, A. S., VARDUND, T., LUND, V., KAPPERUD, G.: Detection of low numbers of pathogenic Yersinia enterocolitica in environmental water and sewage samples by nested polymerase chain reaction. J. Appl. Microbiol. 87, 814–21 (1999). 28. WEYNANTS, V., JADOT, V., DENOEL, P. A., TIBOR, A., LETESSON, J. J.: Detection of Yersinia enterocolitica serogroup O:3 by a PCR method. J. Clin. Microbiol. 34, 1224–7 (1996). 29. WREN, B. W., TABAQCHALI, S.: Detection of pathogenic Yersinia enterocolitica by the polymerase chain reaction [letter] [see comments]. Lancet 336, 693 (1990).
Corresponding author: HOLGER C. SCHOLZ, Institute of Animal Hygiene and Public Veterinary Health, An den Tierkliniken 17, 04103 Leipzig, Germany Tel.: ++49-341-9738165; Fax: ++49-341-9738198; e-mail: [email protected]
A Highly Specific One-step PCR – Assay for the Rapid Discrimination of Enteropathogenic Yersinia enterocolitica from Pathogenic Yersinia pseudotuberculosis and Yersinia pestis THORSTEN ARNOLD1, ANDREAS HENSEL1, RALF HAGEN2, STOJANCA ALEKSIC3, HEINRICH NEUBAUER2 and HOLGER C. SCHOLZ1 1
Institute of Animal Hygiene and Public Veterinary Health, Leipzig, Germany Institut für Mikrobiologie, Sanitätsakademie der Bundeswehr, München, Germany 3 Institute for Hygiene, National Reference Center for Yersiniosis, Hamburg, Germany 2
Received May 25, 2001
Summary Based on differences within the yopT-coding region of Yersinia. enterocolitica, Y. pseudotuberculosis and Y. pestis, a rapid and sensitive one-step polymerase chain reaction assay with high specificity for pathogenic Y. enterocolitica was developed. By this method pathogenic isolates of Y. enterocolitica can be easily identified and discriminated from other members of this genus. The entire coding sequence of the yopT effector gene of Y. pseudotuberculosis Y36 was determined. Key words: Yersinia – yopT – PCR – diagnostic
Introduction Yersinia enterocolitica is a common enteric pathogen responsible for a variety of disorders in humans and animals (NEUBAUER et al., 2001a; NEUBAUER et al., 2001b). Animals are often asymptomatically infected, but do represent a potential source for human infection (FREDRIKSSON-AHOMAA et al., 1999; RAMIREZ et al., 2000; BORCH et al., 1996). When transmitted to humans, pathogenic strains of Y. enterocolitica can cause an acute gastroenteritis, enterocolitis, and mesenteric adenitis, as well as a variety of extra intestinal disorders (TAUXE et al., 1987; HOOGKAMP-KORSTANJE and KONING, 1990; ABDEL-HAQ et al., 2000). Among the six biotypes, and more than 28 serotypes of Y. enterocolitica described to date, only few clones are considered to be pathogenic. Virulence is strictly associated with the presence of the (67–72 kb) Yersinia virulence plasmid (CORNELIS et al., 1998) although non-plasmid carrying Yersinia isolates have been isolated from clinical samples (GRANT et al., 1998). Phenotypic differentiation between pathogenic and non-pathogenic strains of Y. enterocolitica and other Yersinia strains necessitates a complex combination of biochemical and serological tests which have been reported to
provide inconsistent results and to be time consuming (NEUBAUER et al., 2001c; WAAGE et al., 1999; VISHNUBHATLA et al., 2001). Markers for pathogenicity have been attributed to both, the chromosome and the Yersinia virulence plasmid (KWAGA et al., 1992; IBRAHIM et al., 1997; NILSSON et al., 1998). Consequently, various different DNA probes and PCR assays were developed to detect the organism in environmental samples and to differentiate between virulent and avirulent strains. However, so far, both types of markers (chromosomal and plasmid borne) must be used in combination to distinguish pathogenic from non-pathogenic Yersiniae, and Y. enterocolitica from Y. pseudotuberculosis (IBRAHIM et al., 1997; NAKAJIMA et al., 1992). In addition, problems occurred when multiple primer combinations were used in a single PCR reaction (WEYNANTS et al., 1996). Recently, a PCR assay based upon partial 16S rDNA amplification was developed to distinguish between Y. enterocolitica and Y. pseudotuberculosis (NEUBAUER et al., 2000). However, due to cross reactions within the genus Yersiniae, this method requires biochemical characterization of the isolate before PCR. 0723-2020/01/24/02-285 $ 15.00/0
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T. ARNOLD et al.
In this study we describe a specific, sensitive and rapid one-step PCR assay based on the plasmid-located virulence gene yopT to detect plasmid carrying Y. enterocolitica in pure cultures and artificially contaminated feces. The assay is also suitable to differentiate between pathogenic strains of Y. enterocolitica, Y. pseudotuberculosis and Y. pestis.
Material and methods Bacterial strains and growth conditions Y. enterocolitica Y11 (DSM 13030) was obtained from the Deutsche Sammlung von Mikroorganismen, Baunschweig, Germany. Y. pseudotuberculosis Y36, a virulent strain isolated from a guinea pig, was provided by Dr. G. Wolf, LMU Munich. For non-selective cultivation cells were grown in Luria Bertiani (LB) medium. For selective enrichment of Y. enterocolitica from feces, cells were grown in Yersinia selective medium (Oßmer). Inactivated Y. pestis EV76, Burkolderia mallei and B. pseudomallei, Brucella abortus and Francisella tularensis were provided by Dr. Niederwöhrmeier, WIS, Munster, Germany. Further inactivated Y. pestis strains were provided by Dr. Carniel, Institut Pasteur, Paris, France or J. Prior, CBD, Porton Down, UK. Bacteria of these 17 strains (108–109 bacteria/ml) were inactivated by heat before sample preparation. Cloning and sequencing of yopT from Y. pseudotuberculosis Y36 Y. pseudotuberculosis Y36 was grown at 37 °C on Luria Bertiani (LB) agar plates. The primer combination YopT-ff1 (5′ATGGACAGTATTCACGGACAC-3′) and YopT-rr1 (5′-TAAACCTCCTTGGAGTCAAATG-3′) derived from the known yopT coding sequence of Y. enterocolitica (ATCC 9610) was used to amplify the entire coding region of yopT. Single colonies were suspended in 500 µl double deionized water (ddH20). The mixture was heated for 15 min at 94 °C, and placed on ice. After centrifugation (13.000 × g, 5 min), 3 µl of the supernatant were used as template in the PCR assay. The reaction was carried out in a final volume of 50 µl, containing 5 µl 10× PCR-reaction-buffer (Roche, Mannheim, Germany), 5 µl 2mM dNTP mix (Prime Zyme), 10 pmol of each primer, 0.5 U Taq DNA polymerase (Roche, Mannheim), and 3 µl of template DNA. Optimization of cycling conditions resulted in initial denaturation for 5 min at 94 °C followed by 30 cycles each consisting of 80 sec. min denaturation at 94 °C, 40 sec annealing at 59 °C, and 80 sec. min extension at 72 °C, and a final extension step of 7 min. Amplification was performed in a Perkin-Elmer GeneAmp PCR System 2400 (PE, Weiterstadt, Germany). 5 µl of the PCR product was run on a 1%
agarose gel at a constant voltage of 140 V in TAE buffer (40 mM Tris [pH 7.6], 20 mM acetic acid, and 1 mM EDTA). The gel was stained with ethidium bromide and the PCR product was visualized under UV light. The resulting PCR product of 969 bp was directly cloned into the vector pXcmI, exhibiting an T-overhang suitable for direct cloning of amplified PCR products. Its nucleotide sequence was determined. DNA sequencing of the insert was carried out with a LI-COR DNA sequencer model 4000. EMBL nucleotide databank analysis confirmed the presence of yopT in Y. pseudotuberculosis. Generation of Yersinia enterocolitica specific oligonucleotides and partial amplification of yopT fromY. enterocolitica A multiple alignment of the amplified sequence with the yopT coding sequence from Y. enterocolitica (derived from plasmid pYVe227), and Y. pestis (derived from plasmid pCD1) was carried out with ClustalW 1.8. Differences between the yopT coding region of the three species were used to design Y. enterocolitica specific oligonucleotides. The partial alignment of yopT used for the design of Y. enterocolitica specific oligonucleotides is shown in Fig 1. The primer pair yopT-fw1 (5′-TATGTGCACATTGGATTT3′) and yopT-r1 (5′-AATGATACATAGAATTTT-3′) was used to amplify a 478 bp fragment of yopT. Template preparation and detection of the PCR was carried out as described above. Optimization of cycling conditions resulted in initial denaturation for 5 min at 94 °C followed by 35 cycles each consisting of 1 min denaturation at 94 °C, 40 sec annealing at 43 °C, and 1 min extension at 72 °C, and a final extension step of 7 min. Detection of Y. enterocolitica in artificially contaminated feces Three grams of feces were mixed with 3 ml of bacterial suspensions (culture in mid-exponential growth phase) of Y. enterocolitica Y11 (DSM 13030) containing 3 × 109 to 3 × 101 cells and mixed by stirring for 5 min to obtain final concentrations of 109 to 101 bacteria per gram feces. The volume of each sample was adjusted to 10 ml with ringer-solution. 1 ml of this mixture was used for enrichment in either 9 ml of selective (Oßmer) or non-selective (Luria Bertiani) medium. Immediately after inoculation and after 2, 4, 6, and 24 hours of enrichment, 1ml of the suspension was centrifuged at 300 × g in an Eppendorf tube to remove crude particles. The supernatant was centrifuged again at 12.000 × g. The pellet was washed twice in sterile phosphate buffered saline and resolved in a final volume of 200 ml. 10 ml were directly used for the PCR reaction. For direct bacterial DNA-extraction from contaminated feces without foregoing enrichment, the QIAamp DNA Stool Mini Kit (Qiagen) was used.
Fig 1. Alignment of the yopT nucleotide sequence (coding strand) used for the construction of Y. enterocolitica specific oligonucleotides. The oligonucleotides for the specific amplification of yopT in Y. enterocolitica are shown in bold letters. The corresponding sequences of Y. pestis and Y. pseudotuberculosis are given in the alignment below.
PCR based identification and differentiation of Yersinia spp.
Results and Discussion YopT encodes a 35.5 kDa effector protein (YopT) which induces a cytotoxic effect in HeLa cells and macrophages. The effect on HeLa cells consists of disruption of the actin filaments and alteration of the cell cytoskeleton (CORNELIS et al., 1998; IRIARTE and CORNELIS, 1998). In previous studies yopT was detected on the plasmid of pathogenic Y. enterocolitica as well as in the agent of plague, Y. pestis, but was either not detected or reported to be absent from Y. pseudotuberculosis (CORNELIS et al., 1998; HEESEMAN and HENSEL, 2000). In this study the entire yopT coding sequence was proved to be present in Y. pseudotuberculosis Y36 and its sequence was determined. Computer assisted analysis of the sequencing data confirmed the presence of the yopT coding sequence on the plasmid of Y. pseudotuberculosis Y36 (Acc. No., AJ304833). Sequence comparison with Y. enterocolitica and Y. pestis revealed a number of mismatches between the three species. This finding was used to develop a PCR-based assay to specifically detect- and to discriminate pathogenic strains of Y. enterocolitica from Y. pseudotuberculosis and Y. pestis. In order to prove the specificity of the PCR assay, various pathogenic and nonpathogenic strains of Y. enterocolitica and Y. pseudotuberculosis of different serovar/biovar combinations were investigated. The bacterial strains used in this study are listed in Tab. 1. As negative controls, various type strains of Yersinia spp., and members of other bacteria causing similar infections in humans or being possible sample contaminants were included. The yopT specific PCR product of 478 bp was only amplified from DNA of plasmid bearing Y. enterocolitica. No PCR product was obtained with either DNA of other Yersiniae (pathogenic Y. pseudotuberculosis and non pathogenic Yersiniae) or DNA obtained from other bacteria (Tab. 1). Therefore, this assay is highly specific for plasmid bearing Y. enterocolitica. To examine the detection limit for the PCR assay, a series of 10-fold dilutions of Y. enterocolitica-suspension beginning with 108 cells/ml in ddH2O was carried out. A PCR product was yielded still at a concentration of 104 cells when cells were directly used as template without a foregoing proteaseK treatment. To improve the detection limit, cells from each dilution were suspended in lysis buffer containing a final concentration of 0.025% Tween 20 [Sigma], and 0, 2 mg/ml proteinaseK [Merck], and incubated for 1 h at 56 °C. Using these optimized conditions 102 cells were detected. To determine the influence of fecal inhibitors porcine feces were artificially contaminated with different amounts of Y. enterocolitica. During the entire cell preparation procedure no heat treatment was used in order to keep the natural flora of microorganisms of the feces. No PCR product was yielded in the negative control with uninoculated feces demonstrating the specificity of yopTfw1 and yopt-r1. While no PCR product was amplified immediately after inoculation, 2 and 6 h of enrichment in non-selective medium were sufficient to detect Y. enterocolitica with a minimum of 108 and 105 cells/g feces, respectively. After 24 h of enrichment in LB-medium prior
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to PCR, detection of Yersiniae was achieved with 102 cells/g feces (data not shown). Interestingly, the detection limit in selective medium was 104 to 103 cells/g feces, indicating a strong negative effect of selective enrichment. Direct extraction of bacterial DNA from feces (without enrichment) with the QIAmp DNA stool kit resulted in a detection limit of 106 cells/g feces. Therefore, it can be suggested that inhibitory substances are not completely removed during the DNA preparation procedure. The exact bacterial cell number in feces of Yersiniashedding patients is unknown and varies in different stages of infection. However, the low detection limit of 102 cells/g feces may permit the rapid and specific detection of pathogenic Yersina enterocolitica in feces of infected patients within 28 h (24 h of enrichment). The assay is highly discriminating for pathogenic Y. enterocolitica in a single PCR reaction. Rapid detection and discrimination of enteropathogenic strains of Y. enterocolitica from other Yersinia strains as well as from pathogenic members of the genus Enterobacteriaceae is important for diagnostic purposes and for the hygiene control in food production. Most of the commercially available identification systems are time-consuming, and the sensitivity at species level is unsatisfying (WAAGE et al., 1999; LINDE et al., 1999; NEUBAUER et al., 1998; NEUBAUER et al., 2001b; VISHNUBHATLA et al., 2001). The detection of chromosomal virulence markers by PCR like ail, inv or yst (FENG et al., 1992; FENWICK and MURRAY, 1991; IBRAHIM et al., 1997; VISHNUBHATLA et al., 2000; JOURDAN et al., 2000) does not reflect the actual pathogenicity of an isolate. Although the mentioned and various other chromosomally located genes contribute to virulence, in most cases pathogenicity is associated with the presence of the virulence plasmid. Only few virulent isolates lacking known virulence genes (chromosomal and plasmid) have been described (GRANT et al., 1998). In this case the molecular mechanism of virulence is not known, but it can be assumed that the clonality of the isolates used was doubtful. Therefore, it can be postulated that markers specific for the virulence plasmid are most suitable to detect virulent strains. However, the diagnostic value of plasmid-encoded sequences as a diagnostic tool is discussed controversially as the plasmid can be lost during cultivation under laboratory conditions (FENWICK and MURRAY, 1998; IBRAHIM et al., 1992; WREN and TABAQCHALI, 1990). In our investigations, however, we observed that the plasmids of each strain tested were stable for at least 15 subcultivations (data not shown). In routine diagnosis when the pathogen is isolated from different specimens and identified without extensive subcultivation of the microorganism, the loss of the plasmid should not be observed. The sequences used to create Y. enterocolitca specific oligonucleotides might also be useful for the development of Y. pestis and Y. pseudotuberculosis specific PCR assays. In our investigations we could also show that the yopT based PCR assay is capable to specifically detect Y. enterocolitica in artificially contaminated feces in 24 hours with excellent correlation to that of routine bacterial diagnostics (data not shown). Future investigations
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Table 1. Listing of strains used in PCR analyses for partial yopT amplification. Species
serogroup/strain
source
No. of isolates tested
positive for yop T
pathogenic Yersinia strains (p+)1 (p+)2
O:3, O:5,27, O:9, O:2a.2b.3
H3, A4 H, A
21 19
+ –
Y. enterocolitica
(p–)
O:3, O:5 , O:6,30, O:13,7, O:4,33, O:7,8, O:41,43
H, A
92
–
Y. pseudotuberculosis Y. intermedia
(p–)
H, A
29 15
– –
1 1 1 1 1 1 1 1 1 1
– – – – – – – – – –
1 1 1 1 1 1 1 1 1 1
+ – – – – – – – – –
1 1 1 1 1 1 1 1 1 1 1 1 1 1
– – – – – – – – – – – – – –
Y. enterocolitica Y. pseudotuberculosis apathogenic Yersinia strains
O:3, O:5, O:16, O:17, O:40 , O:49,51
Yersinia pestis strains PKH 4519 Turquie 10-1 521 Senegal Thierno 523 Senegal Fay 524 Kenya 147 537 Congo Belge Lita PKR 25 564 Exu 21 567 Con.Bel.Elisab 678 Hamburg 9 695
(p+) (p– ) (p+) (p+) (p+) (p+) (p+) (p+) (p+) (p+)
Kurdistan Turkey Senegal Senegal Kenya Zaire Kurdistan Brazil Zaire Germany
Yersinia type strains Y. enterocolitica subsp. paleartica subsp. enterocolitica Y. pseudotuberculosis Y. intermedia Y. frederiksenii Y. kristensenii Y. aldovae Y. bercovieri Y. rhodei Y. mollaretti
(p+) (p– (p+)
DSM 213030 ATCC 9610 ATCC 29833 ATCC 29909 ATCC 33641 ATCC 33638 ATCC 35236 ATCC 43970 ATCC 43380 ATCC 43969
control group Salmonella enteritidis Salmonella typhimurium Salmonella typhimurium Salmonella senftenberg Enterococcus faecalis Enterobacter aerogenes Escherichia coli Escherichia coli Pseudomonas aeruginosa Staphylococcus aureus Brucella abortus Burkholderia pseudomallei Burkholderia mallei Francisella tularensis 1
p+ – virulence plasmid bearing p– – virulence plasmidless 3 H – human source 4 A – animal source 2
DT 104 SM 5569 775 W D7 / 63 DSM 30053 ATCC 11229 DSM 301 ATCC 15442 DSM 346 ATCC 15682 ATCC 15310
PCR based identification and differentiation of Yersinia spp.
will prove whether the developed assay is also suitable to rapidly detect pathogenic Y. enterocolitica in food or food products and in feces of infected patients. Based on our results we recommend this assay as a simple and time saving method to specifically identify virulence plasmid bearing Y. enterocolitica. Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (GRK-39/2) under project: “Schlachttierbelastung und Produktsicherheit”.
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Corresponding author: HOLGER C. SCHOLZ, Institute of Animal Hygiene and Public Veterinary Health, An den Tierkliniken 17, 04103 Leipzig, Germany Tel.: ++49-341-9738165; Fax: ++49-341-9738198; e-mail: [email protected]