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Comparison of Plasmids of Strains of Yersinia enterocolitica Biovar 1A with the virulence Plasmid of a Pathogenic Y. enterocolitica Strain
Zbl. Bakt. 285, 52-63 (1996) © Gustav Fischer Verlag, Stuttgart· Jena . New York
Comparison of Plasmids of Strains of Yersinia enterocolitica Biovar lA with the virulence Plasmid of a Pathogenic Y. enterocolitica Strain ASTRID LEWIW, ECKHARD STRAUCHl, STEFAN HERTWIGl, BERND HOFFMANN 2 , HERBERT NATTERMANN 2 , and BERND APPELl 1 2
Robert Koch-Institut, Fachbereich Genetik und Gentechnik, Berlin, Germany FU Berlin, Institut fur Mikrobiologie und Tierseuchen des Fachbereichs Veterinarmedizin, Berlin, Germany
Received May 13, 1995 . Revision received January 3, 1996 . Accepted February 23, 1996
Summary The plasmid content of apathogenic Y. enterocolitica biovar lA strains was determined and the plasmids were compared with the virulence plasmid of a pathogenic Y. enterocolitica strain. About 38% of the selected biovar 1A strains contained plasmids of different sizes ranging from 2.7 kb to more than 70 kb. Some of the larger plasmids had a size similar to that of the virulence plasmid of a pathogenic reference strain. The restriction patterns of these plasm ids were different from the restriction pattern of the virulence plasmid of the pathogenic reference strain. Differences were also observed in hybridization studies with the virulence plasmid. The plasmids from 15 out of 16 biovar lA strains showed no homology, whereas the plasmid of one biovar 1A strain partially hybridized to the virulence plasmid.
Introduction Yersinia enterocolitica is an important enteropathogen for humans; it is predominantly transmitted by food, especially by raw meat and meat products (18, 20, 22). Y. enterocolitica infections are the cause of mesenterial, extramesenterial and immunopathological syndromes and can be associated with aseptic secondary disease. Strains of Y. enterocolitica biovar 1A are considered to be apathogenic and can be detected in food, drinking water, milk and vegetables as well as in meat from pigs, cattle, sheep and poultry (3, 9, 16). However, biovar 1A strains were also reported to have been isolated from pathological events (16). Virulence-associated properties of pathogenic strains can also be detected in vitro. Such virulence tests (autoagglutination, calcium-dependent growth at 37°C, hemagglutination, serum resistance, congo red uptake, pyrazinamidase activity, salicin fer-
Plasm ids of Y. enterocolitica biovar 1A
53
mentation and esculin hydrolysis) can be performed under conditions available in routine diagnostic laboratories and provide information on the virulence of a strain. In pathogenic yersiniae, determinants of pathogenicity are localized on a virulence plasmid of about 70kb (about 40 to 48 MDa) (6). Since the possession of a large plasmid is a prerequisite for the pathogenicity of Yersinia strains, several authors have analysed the plasmid content in different Yersinia species including apathogenic strains (10, 12, 14, 15, 19). Such studies revealed that the existence of a large plasmid in a particular strain does not always result in pathogenicity (10, 19,21). As far as the Y. enterocolitica biovar lA strains are concerned, an extensive study on their plasmid content and function is lacking. Because of the ubiquitous distribution of Y. enterocolitica biovar lA in the environment, in food as well as in isolates from clinical events, a more detailed analysis of the characteristics of these strains should be carried out. In this paper, we report the occurrence of plasmids in Y. enterocolitica biovar lA strains isolated from environmental samples, food and animals. We analysed the invitro virulence-associated properties of these strains, their plasmid profiles and the relationship to a virulence plasmid of a Y. enterocolitica 0: 3 biovar 4 strain.
Materials and Methods
Bacterial strains The study was performed with a set of 42 Y. enterocolitica biovar 1A strains isolated from the environment (manure, water; 36 strains), from food (2 strains), or from animals (pig, calf; 4 strains). A pathogenic Y. enterocolitica reference strain (serovar 3, biovar 4) was included in all tests. The strains used are listed in Table 1.
Strain characterization Serotyping: The O-antigen was determined by slide agglutination (1). Absorbed sera were used when necessary. A few strains had a rough phenotype and agglutinated in saline contro!. Biochemical strain characterization: Strain cultivation and species determination were performed as described by Nattermann (1993) and Aleksic and Bockemuhl (1990). The method of Wauters et a!. (1987) was used for biotyping of Y. enterocolitica. Autoagglutination: Cultures were inoculated into two tubes of MR-VP broth (5 g Bacto peptone, 5 g K2HP0 4, 5 g glucose, pH 7.0). One tube was incubated at 36°C and the other at 28 dc. After 24 hand 48 h, the tubes were checked for agglutination, with care being taken not to shake or disturb the sediment at the bottom and along the sides of the tube. Strains were considered as autoagglutinating if they produced a sediment at the bottom and along the sides of the tube at 36°C but not at 28 dc. Strains that agglutinated at both temperatures were classified as rough. Salting-out test: The strains were grown on agar plates for not more than 24 h. One fresh colony was transferred onto a slide with one drop of (NH4hS04 at a concentration of either 0.2, 0.5, 1.0, or 2.0 M. The strains were classified as positive for the salting-out reaction if an aggregation could be observed within 60s with (NH4h S04 at concentrations of 0.2 and/or 0.5 M. If an aggregation only occurred at concentrations of 1.0 and / or 2.0 M, the strains were classified as negative in the salting-out test. Serum resistance: Bacteria from an agar plate were transferred to liquid medium and incubated at 30°C for 18 h. An aliquot of this culture was diluted with 2 mL of fresh medium and cultivated at 30°C for about 2 h. 0.01 mL of this culture was transferred to 5 mL
54
A. Lewin et al.
Table 1. Bacterial strains strain number
serovar
biovar
origin
Y. e. #4 Y. e. #14 Y. e. #18 Y. e. #40 Y. e. #42 Y. e. #44 Y. e. #56 Y. e. #66 Y. e. #78 Y. e. #102 Y. e. #118 Y. e. #132 Y. e. #157 Y. e. #165 Y. e. #177 Y. e. #263 Y. e. #266 Y. e. #282 Y. e. #348 Y. e. #353 Y.e. #390 Y.e. #392 Y. e. #398 Y. e. #399 Y. e. #412 Y. e. #428 Y. e. #436 Y. e. #454 Y. e. #455 Y. e. #491 Y. e. #519 Y. e. #540 Y.e. #571 Y. e. #657 Y.e. #W3 Y.e. #W10 Y. e. #29845 Y. e. #29854 Y.e. #29807 Y. e. #29881 Y. e. #11233 Y. e. #15673 Y. e. #13169 Y. ps. #29827
0:50,51 0:6,30 0:6,30 0:13,7 0:6,30 0:6,30 n.d. 0:13,7 0:13,7 0:13,7 0:6,31 0:7,8 0:6,30 0:13,7 0:6,30 0:41,43 0:10 n.d. 0:7,8 0:7,8 0:7,8 0:5 0:7,8 0:6,30 0:41,43 0:5 n.d. 0:5 0:5 0:6,31 0:5 0:19,8 0:5 0:5 0:13,7 0:10 0:5 0:5 0:5 0:5 0:7,8 0:5 0:3
1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 4
stream manure manure nver manure manure stream river stream stream stream reservoir reservOir reservOir reservoir reservoir reservOir reservoir reservoir water works reservoir reservoir reservOir reservOir reservoir manure reservoir manure manure reservoir manure reservOir manure manure water works water works food food pig, lung pig, mouth calf (aborted), liver calf (aborted), abomasum pig, rectum wild boar, tonsil
I
plasmid content
+ +
+
+
+
+ + + + + + + + + + + + +
Y. e.: Y. enterocolitica; Y. ps.: Y. pseudotuberculosis; n. d.: not determined; +: plasmid carrying strain: -: strain without plasmids; strains #4 to #W10 were isolated by Dr. Feuerpfeil, Umweltbundesamt, Bad Elster.
Plasmids of Y. enterocolitica biovar lA
55
of physiological NaCI solution. 2 mL of the suspension were plated onto dextrose-agar. Serum from sheep, horse, chicken or beaver which had been purified by centrifugation and sterilised by filtration was dropped onto the dextrose agar with the Yersinia strains. Serum that had been inactivated by heating (56°C, 30 min) was also dropped onto the plate as a control. Strains were classified as serum-resistant if they did not show any growth inhibition in the area with the non-inactivated serum. Low calcium response: Calcium-dependent growth was tested on magnesium oxalate agar as described by Heesemann and Laufs (1983). Congo red uptake. The Yersinia strains were cultivated on plates at 30°C for 24 hand then streaked onto Congo red agar (15 g pancreatic casein,S g peptone from bull testis,S g NaC!, 15g agar-agar, made up to 1000mL with water, together with 50mg congo red in 50 mL phospate buffer pH 7). These agar plates were incubated at 37°C for 24 h. Red colonies were classified as positive for congo red uptake, whereas white or only lightly-coloured colonies were classified as negative.
Plasmid profile Plasmid DNA was extracted according to the method of Kado and Liu (1981). The plasmids were separated by migration in a 0.8% agarose gel in TBE buffer.
Preparation of plasmid DNA Plasmid DNA was extracted from 200 mL cultures of Yersinia by a procedure based on the alkaline lysis method of Birnboim and Doly (4, 5). The cells were resuspended in buffer Pl (50mM Tris/HCl, 10mM EDTA, 100mglmL RNase, pH 8.0), lysed in buffer P2 (200mM NaOH, 1 % SDS) and the lysate was neutralised in buffer P3 (3.0M potassium acetate, pH 5.5). After centrifugation, the DNA in the supernatant was precipitated with isopropanol. The DNA pellet was dissolved in TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8.0). Finally, the plasmid DNA was separated from chromosomal DNA by equilibrium centrifugation in CsCI-ethidium bromide gradients as described in Maniatis et al. (1982).
Plasmid analysis (restriction, hybridization) Restriction analysis of plasmids and agarose gel electrophoresis were performed according to standard procedures (13). Hybridisations with fluorescein-labelled DNA-probes were made with the "Renaissance kit" from Dupont according to the manufacturers' recommendations. DNA in agarose gels was transferred to a nylon membrane (Hybond TM N+). Hybridizations were performed at 65°C in 5 x SSC, 0.1 % SDS, 0.5% blocking reagent, 5% dextran sulfate. Material that did not hybridize to the membrane-bound DNA was washed off at 65°C first with 2 x SSC, 0.1 % SDS and then with 0.2 x SSC, 0.1 % SDS. Results
Occurrence of plasmids in Y. enterocolitica biovar lA strains In order to determine the frequency of the occurrence of plasmids in biovar 1A strains, plasmid profiles of all the strains listed in Table 1 were visualised, together with the virulence plasmid of a pathogenic 0: 3, biovar 4 strain and the virulence plasmid of a pathogenic Y. pseudotuberculosis strain by the method of Kado and Liu (1981). The 42 Y. enterocolitica biovar 1A strains showed the following distribution of plasmids: 26 strains did not contain plasmids while 16 strains (38%) did. The plasmid content of the strains is shown in Table 1. All habitats from which biovar 1A strains had been obtained (river, stream, reservoir, water works, manure, food, animals) revealed strains with plasmids. Fig. 1 shows the plasmid profile of the plasmid-carrying strains
Fig. 1. Plasmid profiles of Y. enterocolitica biovar 1A strains (#56 to #15673), a pathogenic Y. pseudotuberculosis strain (#29827), a pathogenic Y. enterocolitica strain (#13169), and a virulence plasmid-cured derivative of strain #13169 (#13169/2).
'""
~
rt
(b
5'
~
(b
r-'
?>
0\
Plasmids of Y. enterocolitica biovar 1A
57
as well as the plasmid profile of two different pathogenic strains. The plasmids could be classified according to their size: small plasmids migrating below the chromosomal DNA and large plasmids migrating above or together with the chromosomal DNA (see Fig. 1). Some of the large plasm ids migrated to about the same position in the gel as did the virulence plasmids of the pathogenic reference strains. Properties of plasmid-containing biovar lA strains
To characterize the plasmid-containing representatives of biovar lA strains with respect to possible pathogenic features, we tested them for the following in-vitro virulence-associated reactions: esculin hydrolysis, salicin fermentation, pyrazinamidase, lecithinase, lipase, autoagglutination, salting-out, low calcium response, congo red uptake, and serum resistance. Apathogenic Yersinia strains are usually positive for esculin hydrolysis, salicin fermentation, pyrazinamidase, lecithinase, and lipase, whereas they are usually negative for autoagglutination, salting-out, low calcium response, congo red uptake, and serum resistance. Table 2 presents an overview of the in-vitro virulence-associated properties of the strains. The selected strains which had been classified as biovar lA strains by routine diagnostic methods did not in all cases show the typical behaviour assigned to apathogenic strains of Y. enterocolitica biovar lA. Some of the 16 strains showed one or several in-vitro virulence-associated properties; 4 strains autoagglutinated; after 48 h, all
Table 2. Physiological properties of plasmid-containing Y. entercolitica biovar 1A strains and a pathogenic Y. enterocolitica strain esc
sal
pyr
lip
lec
aa
56 + 78 + 353 + 398 +/428 + 491 + 519 + 571 + 657 + W3 + 29807 + 29881 + 29845 + 29854 + 11233 + 15673 +/13169 -
+ + + +/+
+ + + +/+ + + + + + + + + + + +
+ + + +/+ + + + +/+ + + + + + +/-
+ +
+/-
strain
+ + + + + + + + + +/-
n.d. -
n.d. -
+/+ + + + + + + + + + +/-
salt
Icr
cr (b)
+
+ (+) (+) + (+) + (+) (+) (+) + (+) (+) (+) (+) (+) (+) +
+
+/-
+/-
cr (a)
+
+
+
+/-
+
+
+
ser (a)
ser (b)
ser (c)
ser (d)
+
+ + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + +
+ +
+ + + +
+ +
esc: esculin hydrolysis; sal: salicin fermentation; pyr = pyrazinamidase; lip = lipase; lec = lecithinase; aa = autoagglutination; salt = salting-out; Icr = low calcium response; cr = congo red-uptake (a: after 24 h, b: after 48 h); ser = serum resistance (serum from: a: sheep, b: horse, c: chicken, d: beaver); +/-: different reactions in repeated tests, (+): weak positive reaction.
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A. Lewin et al.
strains showed at least a weak positive reaction for congo red uptake, 2 strains were positive for the salting-out reaction and all strains were resistant against at least 2 of the 4 sera used. None of the biovar 1A strains was positive for the low calcium response reaction, which is known to be plasmid-encoded in pathogenic strains.
Size of plasmids of biovar lA strains In order to determine the size of some representative plasmids, restriction enzyme analysis was performed and the molecular weight of the plasmids was calculated on the basis of the size of the restriction fragments generated. For plasmid size determination we selected representatives from different habitats (environment, food, clinical isolates) containing plasmids migrating above the chromosomal DNA and three strains containing a small plasmid migrating below the chromosomal DNA. The virulence plasmid of a pathogenic strain was used as a reference. The results are shown in Table 3. The small plasmids had sizes between 2.7 and 6.3 kb. The large plasmids were of sizes ranging between about 50 and 70 kb.
Restriction patterns of plasmids of biovar lA strains The existence of large plasmids in biovar 1A strains indicated that there might be some similarity between these plasmids and the virulence plasmid. To answer this question, we compared the restriction patterns of a virulence plasmid with the patterns of several large plasmids of biovar 1A strains. The strains were chosen as representatives of the different habitats used for strain isolation (environmental isolate from a stream, food isolate, clinical isolate). Fig. 2 shows the patterns for the restriciton enzymes EcoRI, HindIII, and BamHI. The restriction patterns of the plasmids of the biovar 1A strains were different from the pattern of the virulence plasmid for all enzymes tested. These results do not point to a high degree of structural relationship between the virulence plasmid and the selected plasmids in biovar 1A strains.
Homology between the virulence plasmid and plasmids of biovar lA strains To obtain more information concerning possible similarities between the respective DNA sequences, we performed Southern blots with consecutive hybridization between the plasmids of the biovar 1A strains and the virulence plasmid of strain #13169. Table 3. Sizes of selected plasmids of Y. enterocolitica biovar 1 A strains compared with a virulence plasmid (p13169) strain #
estimated plasmid size (kb)
#13169
75
#78 #15673 #29845
52 65 70
#519 #29807 #29881
4.3 2.7 6.3
Plasmids of Y. enterocolitica biovar 1A
59
kb 23.1 9.4 6.6
4.4
2.3 2.0
0.6
EcoRI
HindlH
BamHI
Fig. 2. Comparison of restriction patterns of plasmids of Y. enterocolitica biovar 1A (#78, #15673, #29845) to a virulence plasmid (#13169).
The plasmids of 16 strains of Y. enterocolitica biovar 1A were digested with restriction enzymes, the fragments were separated by gel electrophoresis, transferred to a nylon membrane and hybridized with the labelled virulence plasmid of strain #13169. While the plasmids from 15 out of the 16 strains did not hybridize, the plasmid of strain #15673 showed some homology to the virulence plasmid indicating a corresponding similarity between the plasmid of the biovar 1A strain #15673 and the virulence plasmid of strain #13169. Fig. 3 shows the homology between the plasmid of strain #15673 and the virulence plasmid of strain #13169. Discussion
Y. enterocolitica biovar 1A strains, which are ubiquitously distributed and can be found in soil, water and food, are usually considered to be apathogenic. They can, however, sometimes be found in clinical isolates. This observation - together with the observation that biovar 1A strains sometimes contain large plasmids - raises the question as to whether the epidemiological importance of these strains may be underestimated. To answer this question, we analysed a collection of biovar 1A strains for their plasmid content and their in-vitro virulence-associated properties. Furthermore, the plasmids were examined for a possible relationship to a typical virulence plasmid of a pathogenic Y. enterocolitica strain.
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A. Lewin et al.
Fig. 3. Homology between the virulence plasmid of Y. enterocolitica #13169 to the virulence plasmid of Y. pseudotuberculosis #29827 and to the plasmid of the Y. enterocolitica biovar 1A strain #15673. The plasmids were digested with the restriction enzyme HindIII and the fragments were separated in an agarose gel (A). The DNA was transferred from the gel to a membrane and hybridized with the fluorescein-labelled virulence plasmid of strain #13169 (B).
In a first step, biotyping was performed by routine diagnostic methods. 42 strains which could be classified as biovar 1A by these reactions were selected for analysis of their plasmid content. Our findings are in agreement with the data of other authors (e. g. 10,14,15) showing that plasmids are widely distributed in Yersinia strains and are not restricted to pathogenic strains. 38% of the selected biovar 1A strains carried plasmids most of which migrated to about the same position in an agarose gel as did the virulence plasmid under standard conditions. In routine diagnostic laboratories, these plasmids might thus be classified as virulence plasmids. The size of selected large plasmids was determined to be between 50 and 70 kb, whereas the size of the selected small plasmids was estimated to be between 2.7kb and 6.3kb.
Plasmids of Y. enterocolitica biovar 1A
61
Since these results raise the question whether in particular the large plasmids might carry some pathogenicity factors, we tested the strains for in-vitro virulence-associated characteristics. None of the strains classified as biovar lA was positive for the low calcium response reaction. However, many of the strains indeed possess some other virulence-associated characteristics as shown in Table 2. This result can be interpreted in two ways: Either the biovar lA strains have some virulence-associated characteristics and thus some pathogenic potential, or the in-vitro tests usually performed for the identification of pathogenic strains are not suitable for the biovar lA strains. The fact that biovar lA strains can also be isolated in connection with pathological events (16) supports the first hypothesis. On the other hand, the possibility that the tests for virulence-associated properties usually performed do not allow a reliable assessment of the virulence of biovar lA strains is in agreement with the observations of other authors (11). Both possibilities point to the necessity of applying methods different from the commonly used biochemical tests in order to determine the pathogenicity of Yersinia strains. Furthermore, the relevance of the existence of large plasmids in these strains should be investigated. These conclusions are in agreement with the results of Wachsmuth et al. (1984), who found that routine or simple plasmid profile analysis has a limited application in the diagnosis and epidemiology of infections caused by yersiniae. For these reasons, we applied molecular techniques for a further characterisation of the plasmids detected in our work. A comparison of the restriction patterns of selected large plasmids with the restriction pattern of a virulence plasmid showed that there were no similarities on this level. Work is in progress to analyse the plasmids of biovar lA strains to find out whether these plasm ids are related to each other or can be divided into subgroups. In most cases our hybridizations of the complete virulence plasmid to plasmids of biovar lA strains detected no homology at all. Interestingly, one strain (#15673) showed some homology to the virulence plasmid. Plasmid p15673 has a size of about 65 kb and thus a size similar to that of the virulence plasmids of pathogenic Yersinia strains. The restriction pattern of p15673, however, does not resemble the restriction pattern of the virulence plasmid. We are now performing further hybridizations to determine exactly which regions of the virulence plasmid are homologous to the plasmid of strain #15673. Preliminary results have localized the homologous region to sequences of the virulence plasmid which are outside the conserved calcium-regulation region (data not shown). It is noteworthy that strain #15673 is a clinical isolate (from the abomasum from an aborted calf foetus) and in addition has some in-vitro virulence-associated characteristics. This may indicate that the determination of a possible virulence of strains able to persist within organs requires virulence tests which are different from those commonly used. The homology between the virulence plasmid and the plasmid of biovar lA strain #15673 raises the question as to whether the homologous regions carry any virulence-associated genes, and whether these regions provide any potential for pathogenicity in such biovar lA strains. Our future work will concentrate on the analysis of the homologous sequences of p15673 and of the virulence plasmid in order to determine their biological function and their clinical importance. Acknowledgements. We thank B. Karbowiak and S. Wolgast-Hoffmann for excellent technical assistance. We also thank Dr. Feuerpfeil, Dr. Stephan, and D. Knabner for isolating and providing bacterial strains.
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A. Lewin et al. References
1. Aleksic, S. and J. Bockemuhl: Proposed revision of the Wauters et al. antigenic scheme for serotyping of Yersinia enterocolitica. J. Clin. Microbiol. 20 (1984) 843-845 2. Aleksic, S. and J. Bockemuhl: Mikrobiologie und Epidemiologie der Yersiniosen. Immun.Infekt. 18 (1990) 178-185 3. Aulisio, C. C. G., W. E. Hill, J. T. Stanfield, and R. L. jr. Sellers: Evaluation of virulence factor testing and characteristics of pathogenicity in Yersinia enterocolitica. Infect. Immunol. 40 (1983) 330-335 4. Birnboim, H. A rapid alkaline extraction method for the isolation of plasmid DNA. Meth. Enzymol. 100 (1983) 243-255 5. Birnboim, H. C. andJ. Doly: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7 (1979) 1513-1522 6. Heesemann, J.: Enteropathogene Yersinien: Pathogenitatsfaktoren und neue diagnostische Methoden. Immun. Infekt. 18 (1990) 186-191 7. Heesemann, J. and R. Laufs: Construction of a mobilizable Yersinia enterocolitica virulence plasmid. J. Bacteriol. 155 (1983) 761-767 8. Kado, C. I. and S. T. Liu: Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol. 145 (1981) 1365-1373 9. Karib, H. und H. Seeger: Vorkommen von Yersinien- und Campylobacter-Arten in Lebensmitteln. Fleischwirtsch. 74 (1994) 1104-1106 10. Kleemann, J.: Untersuchungen zum Vorkommen und Verhalten von Yersinia enterocolitica in Rohwurst. Diss., Berlin, 1993 11. Kleemann, J. und T. Bergann: Yersinia spp. in frischer Rohwurst. Fleischwirtsch. 74 (1994) 1101-1103 12. Kuznetzov V. G., V. N. Bagriantsev, and V. M. Kitaev: Vydelenie shtammov Yersinia intermedia s plazmidoi molekuiarnoi massy 82 mega dalton v prirodnykh pohuliatsiiakh iersinii. (The isolation of Yersinia intermedia strains with a plasmid of 82-megadalton molecular weight in natural populations of Yersinia). Zhurnal Mikrobiologii, Epidemiologii i Immunobiologii 11 (1990) 12-16 13. Maniatis, T., E. F. Fritsch, and J. Sambrook: Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1982 14. Marranzano, M., A. Agodi, and M. Gulisano: Molecular characterization of Yersinia strains isolated from human and environmental sources. Microbiologica 16 (1993) 5762 15. Mingrone, M. G. and M. Fantasia: Characteristics of Yersinia spp. isolated from wild and zoo animals. J. Wildlife Dis. 24 (1988) 25-29 16. Nattermann, H.: Untersuchungen zur Diagnostik, Epizootiologie, Pathogenese und Klinik der Yersinia enterocolitica-Infektion bei Schweinen, Schafen, Hiihnern und Hunden. Vet.-Med. Diss. B, HU Berlin (1986) 17. Nattermann, H.: Diagnose von Yersinia enterocolitica. Mitteilung 111993 der 12. AVIDTagung am 24. und 25. Juni 1993 in Berlin, Anlage VIII S.l 18. Nattermann, H., F. Horsch, K. Maier und D. Beutling: Neue Erkenntnisse zur Yersinia enterocolitica-Infektion des Schweines. Deutsche Veterinarmedizinische Gesellschaft, Tagung der Fachgruppe "Bakteriologie und bakterielle Krankheiten" Schloss Rauischholzhausen bei Marburg, 8.-10 Juni 1994, Abstr. in: Tagungsband S. 251-259 19. Prpic, J. K., R. M. Robins-Browne, and R. B. Davey: In vitro assessment of virulence in Yersinia enterocolitica and related species. J. Clin. Microbiol. 22 (1985) 105-110 20. Tauxe, R. V., J. Vandepitte, G. Wauters, S. M. Martin, V. Goossens, P. De Mol, R. van Noyer, and G. Thiers: Yersinia enterocolitica infections and pork: the missing link. Lancet 1 (1987) 1129-1132 21. Wachsmuth, K., B. A. Kay, and K. A. Birkness: Diagnostic value of plasmid analyses and assays for virulence in Yersinia enterocolitica. Diagn. Microbiol. Infect. Dis. 2 (1984) 219-228
c.:
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22. Wauters, G.: Carriage of Yersinia enterocolitica serotype 3 by pigs as a source of human infection. Contr. Microbiol. Immunol. 5 (1979) 249-252
23. Wauters, G., K. Kandala, and M. Janssens: Revised biogrouping scheme of Yersinia enterocolitica. Cont. Microbiol. Immunol. 9 (1987) 14-21 Prof. Dr. Bernd Appel, Robert Koch-Institut, Fachbereich 5, Nordufer 20, D-13353 Berlin, Germany
Comparison of Plasmids of Strains of Yersinia enterocolitica Biovar lA with the virulence Plasmid of a Pathogenic Y. enterocolitica Strain ASTRID LEWIW, ECKHARD STRAUCHl, STEFAN HERTWIGl, BERND HOFFMANN 2 , HERBERT NATTERMANN 2 , and BERND APPELl 1 2
Robert Koch-Institut, Fachbereich Genetik und Gentechnik, Berlin, Germany FU Berlin, Institut fur Mikrobiologie und Tierseuchen des Fachbereichs Veterinarmedizin, Berlin, Germany
Received May 13, 1995 . Revision received January 3, 1996 . Accepted February 23, 1996
Summary The plasmid content of apathogenic Y. enterocolitica biovar lA strains was determined and the plasmids were compared with the virulence plasmid of a pathogenic Y. enterocolitica strain. About 38% of the selected biovar 1A strains contained plasmids of different sizes ranging from 2.7 kb to more than 70 kb. Some of the larger plasmids had a size similar to that of the virulence plasmid of a pathogenic reference strain. The restriction patterns of these plasm ids were different from the restriction pattern of the virulence plasmid of the pathogenic reference strain. Differences were also observed in hybridization studies with the virulence plasmid. The plasmids from 15 out of 16 biovar lA strains showed no homology, whereas the plasmid of one biovar 1A strain partially hybridized to the virulence plasmid.
Introduction Yersinia enterocolitica is an important enteropathogen for humans; it is predominantly transmitted by food, especially by raw meat and meat products (18, 20, 22). Y. enterocolitica infections are the cause of mesenterial, extramesenterial and immunopathological syndromes and can be associated with aseptic secondary disease. Strains of Y. enterocolitica biovar 1A are considered to be apathogenic and can be detected in food, drinking water, milk and vegetables as well as in meat from pigs, cattle, sheep and poultry (3, 9, 16). However, biovar 1A strains were also reported to have been isolated from pathological events (16). Virulence-associated properties of pathogenic strains can also be detected in vitro. Such virulence tests (autoagglutination, calcium-dependent growth at 37°C, hemagglutination, serum resistance, congo red uptake, pyrazinamidase activity, salicin fer-
Plasm ids of Y. enterocolitica biovar 1A
53
mentation and esculin hydrolysis) can be performed under conditions available in routine diagnostic laboratories and provide information on the virulence of a strain. In pathogenic yersiniae, determinants of pathogenicity are localized on a virulence plasmid of about 70kb (about 40 to 48 MDa) (6). Since the possession of a large plasmid is a prerequisite for the pathogenicity of Yersinia strains, several authors have analysed the plasmid content in different Yersinia species including apathogenic strains (10, 12, 14, 15, 19). Such studies revealed that the existence of a large plasmid in a particular strain does not always result in pathogenicity (10, 19,21). As far as the Y. enterocolitica biovar lA strains are concerned, an extensive study on their plasmid content and function is lacking. Because of the ubiquitous distribution of Y. enterocolitica biovar lA in the environment, in food as well as in isolates from clinical events, a more detailed analysis of the characteristics of these strains should be carried out. In this paper, we report the occurrence of plasmids in Y. enterocolitica biovar lA strains isolated from environmental samples, food and animals. We analysed the invitro virulence-associated properties of these strains, their plasmid profiles and the relationship to a virulence plasmid of a Y. enterocolitica 0: 3 biovar 4 strain.
Materials and Methods
Bacterial strains The study was performed with a set of 42 Y. enterocolitica biovar 1A strains isolated from the environment (manure, water; 36 strains), from food (2 strains), or from animals (pig, calf; 4 strains). A pathogenic Y. enterocolitica reference strain (serovar 3, biovar 4) was included in all tests. The strains used are listed in Table 1.
Strain characterization Serotyping: The O-antigen was determined by slide agglutination (1). Absorbed sera were used when necessary. A few strains had a rough phenotype and agglutinated in saline contro!. Biochemical strain characterization: Strain cultivation and species determination were performed as described by Nattermann (1993) and Aleksic and Bockemuhl (1990). The method of Wauters et a!. (1987) was used for biotyping of Y. enterocolitica. Autoagglutination: Cultures were inoculated into two tubes of MR-VP broth (5 g Bacto peptone, 5 g K2HP0 4, 5 g glucose, pH 7.0). One tube was incubated at 36°C and the other at 28 dc. After 24 hand 48 h, the tubes were checked for agglutination, with care being taken not to shake or disturb the sediment at the bottom and along the sides of the tube. Strains were considered as autoagglutinating if they produced a sediment at the bottom and along the sides of the tube at 36°C but not at 28 dc. Strains that agglutinated at both temperatures were classified as rough. Salting-out test: The strains were grown on agar plates for not more than 24 h. One fresh colony was transferred onto a slide with one drop of (NH4hS04 at a concentration of either 0.2, 0.5, 1.0, or 2.0 M. The strains were classified as positive for the salting-out reaction if an aggregation could be observed within 60s with (NH4h S04 at concentrations of 0.2 and/or 0.5 M. If an aggregation only occurred at concentrations of 1.0 and / or 2.0 M, the strains were classified as negative in the salting-out test. Serum resistance: Bacteria from an agar plate were transferred to liquid medium and incubated at 30°C for 18 h. An aliquot of this culture was diluted with 2 mL of fresh medium and cultivated at 30°C for about 2 h. 0.01 mL of this culture was transferred to 5 mL
54
A. Lewin et al.
Table 1. Bacterial strains strain number
serovar
biovar
origin
Y. e. #4 Y. e. #14 Y. e. #18 Y. e. #40 Y. e. #42 Y. e. #44 Y. e. #56 Y. e. #66 Y. e. #78 Y. e. #102 Y. e. #118 Y. e. #132 Y. e. #157 Y. e. #165 Y. e. #177 Y. e. #263 Y. e. #266 Y. e. #282 Y. e. #348 Y. e. #353 Y.e. #390 Y.e. #392 Y. e. #398 Y. e. #399 Y. e. #412 Y. e. #428 Y. e. #436 Y. e. #454 Y. e. #455 Y. e. #491 Y. e. #519 Y. e. #540 Y.e. #571 Y. e. #657 Y.e. #W3 Y.e. #W10 Y. e. #29845 Y. e. #29854 Y.e. #29807 Y. e. #29881 Y. e. #11233 Y. e. #15673 Y. e. #13169 Y. ps. #29827
0:50,51 0:6,30 0:6,30 0:13,7 0:6,30 0:6,30 n.d. 0:13,7 0:13,7 0:13,7 0:6,31 0:7,8 0:6,30 0:13,7 0:6,30 0:41,43 0:10 n.d. 0:7,8 0:7,8 0:7,8 0:5 0:7,8 0:6,30 0:41,43 0:5 n.d. 0:5 0:5 0:6,31 0:5 0:19,8 0:5 0:5 0:13,7 0:10 0:5 0:5 0:5 0:5 0:7,8 0:5 0:3
1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 4
stream manure manure nver manure manure stream river stream stream stream reservoir reservOir reservOir reservoir reservoir reservOir reservoir reservoir water works reservoir reservoir reservOir reservOir reservoir manure reservoir manure manure reservoir manure reservOir manure manure water works water works food food pig, lung pig, mouth calf (aborted), liver calf (aborted), abomasum pig, rectum wild boar, tonsil
I
plasmid content
+ +
+
+
+
+ + + + + + + + + + + + +
Y. e.: Y. enterocolitica; Y. ps.: Y. pseudotuberculosis; n. d.: not determined; +: plasmid carrying strain: -: strain without plasmids; strains #4 to #W10 were isolated by Dr. Feuerpfeil, Umweltbundesamt, Bad Elster.
Plasmids of Y. enterocolitica biovar lA
55
of physiological NaCI solution. 2 mL of the suspension were plated onto dextrose-agar. Serum from sheep, horse, chicken or beaver which had been purified by centrifugation and sterilised by filtration was dropped onto the dextrose agar with the Yersinia strains. Serum that had been inactivated by heating (56°C, 30 min) was also dropped onto the plate as a control. Strains were classified as serum-resistant if they did not show any growth inhibition in the area with the non-inactivated serum. Low calcium response: Calcium-dependent growth was tested on magnesium oxalate agar as described by Heesemann and Laufs (1983). Congo red uptake. The Yersinia strains were cultivated on plates at 30°C for 24 hand then streaked onto Congo red agar (15 g pancreatic casein,S g peptone from bull testis,S g NaC!, 15g agar-agar, made up to 1000mL with water, together with 50mg congo red in 50 mL phospate buffer pH 7). These agar plates were incubated at 37°C for 24 h. Red colonies were classified as positive for congo red uptake, whereas white or only lightly-coloured colonies were classified as negative.
Plasmid profile Plasmid DNA was extracted according to the method of Kado and Liu (1981). The plasmids were separated by migration in a 0.8% agarose gel in TBE buffer.
Preparation of plasmid DNA Plasmid DNA was extracted from 200 mL cultures of Yersinia by a procedure based on the alkaline lysis method of Birnboim and Doly (4, 5). The cells were resuspended in buffer Pl (50mM Tris/HCl, 10mM EDTA, 100mglmL RNase, pH 8.0), lysed in buffer P2 (200mM NaOH, 1 % SDS) and the lysate was neutralised in buffer P3 (3.0M potassium acetate, pH 5.5). After centrifugation, the DNA in the supernatant was precipitated with isopropanol. The DNA pellet was dissolved in TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8.0). Finally, the plasmid DNA was separated from chromosomal DNA by equilibrium centrifugation in CsCI-ethidium bromide gradients as described in Maniatis et al. (1982).
Plasmid analysis (restriction, hybridization) Restriction analysis of plasmids and agarose gel electrophoresis were performed according to standard procedures (13). Hybridisations with fluorescein-labelled DNA-probes were made with the "Renaissance kit" from Dupont according to the manufacturers' recommendations. DNA in agarose gels was transferred to a nylon membrane (Hybond TM N+). Hybridizations were performed at 65°C in 5 x SSC, 0.1 % SDS, 0.5% blocking reagent, 5% dextran sulfate. Material that did not hybridize to the membrane-bound DNA was washed off at 65°C first with 2 x SSC, 0.1 % SDS and then with 0.2 x SSC, 0.1 % SDS. Results
Occurrence of plasmids in Y. enterocolitica biovar lA strains In order to determine the frequency of the occurrence of plasmids in biovar 1A strains, plasmid profiles of all the strains listed in Table 1 were visualised, together with the virulence plasmid of a pathogenic 0: 3, biovar 4 strain and the virulence plasmid of a pathogenic Y. pseudotuberculosis strain by the method of Kado and Liu (1981). The 42 Y. enterocolitica biovar 1A strains showed the following distribution of plasmids: 26 strains did not contain plasmids while 16 strains (38%) did. The plasmid content of the strains is shown in Table 1. All habitats from which biovar 1A strains had been obtained (river, stream, reservoir, water works, manure, food, animals) revealed strains with plasmids. Fig. 1 shows the plasmid profile of the plasmid-carrying strains
Fig. 1. Plasmid profiles of Y. enterocolitica biovar 1A strains (#56 to #15673), a pathogenic Y. pseudotuberculosis strain (#29827), a pathogenic Y. enterocolitica strain (#13169), and a virulence plasmid-cured derivative of strain #13169 (#13169/2).
'""
~
rt
(b
5'
~
(b
r-'
?>
0\
Plasmids of Y. enterocolitica biovar 1A
57
as well as the plasmid profile of two different pathogenic strains. The plasmids could be classified according to their size: small plasmids migrating below the chromosomal DNA and large plasmids migrating above or together with the chromosomal DNA (see Fig. 1). Some of the large plasm ids migrated to about the same position in the gel as did the virulence plasmids of the pathogenic reference strains. Properties of plasmid-containing biovar lA strains
To characterize the plasmid-containing representatives of biovar lA strains with respect to possible pathogenic features, we tested them for the following in-vitro virulence-associated reactions: esculin hydrolysis, salicin fermentation, pyrazinamidase, lecithinase, lipase, autoagglutination, salting-out, low calcium response, congo red uptake, and serum resistance. Apathogenic Yersinia strains are usually positive for esculin hydrolysis, salicin fermentation, pyrazinamidase, lecithinase, and lipase, whereas they are usually negative for autoagglutination, salting-out, low calcium response, congo red uptake, and serum resistance. Table 2 presents an overview of the in-vitro virulence-associated properties of the strains. The selected strains which had been classified as biovar lA strains by routine diagnostic methods did not in all cases show the typical behaviour assigned to apathogenic strains of Y. enterocolitica biovar lA. Some of the 16 strains showed one or several in-vitro virulence-associated properties; 4 strains autoagglutinated; after 48 h, all
Table 2. Physiological properties of plasmid-containing Y. entercolitica biovar 1A strains and a pathogenic Y. enterocolitica strain esc
sal
pyr
lip
lec
aa
56 + 78 + 353 + 398 +/428 + 491 + 519 + 571 + 657 + W3 + 29807 + 29881 + 29845 + 29854 + 11233 + 15673 +/13169 -
+ + + +/+
+ + + +/+ + + + + + + + + + + +
+ + + +/+ + + + +/+ + + + + + +/-
+ +
+/-
strain
+ + + + + + + + + +/-
n.d. -
n.d. -
+/+ + + + + + + + + + +/-
salt
Icr
cr (b)
+
+ (+) (+) + (+) + (+) (+) (+) + (+) (+) (+) (+) (+) (+) +
+
+/-
+/-
cr (a)
+
+
+
+/-
+
+
+
ser (a)
ser (b)
ser (c)
ser (d)
+
+ + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + +
+ +
+ + + +
+ +
esc: esculin hydrolysis; sal: salicin fermentation; pyr = pyrazinamidase; lip = lipase; lec = lecithinase; aa = autoagglutination; salt = salting-out; Icr = low calcium response; cr = congo red-uptake (a: after 24 h, b: after 48 h); ser = serum resistance (serum from: a: sheep, b: horse, c: chicken, d: beaver); +/-: different reactions in repeated tests, (+): weak positive reaction.
58
A. Lewin et al.
strains showed at least a weak positive reaction for congo red uptake, 2 strains were positive for the salting-out reaction and all strains were resistant against at least 2 of the 4 sera used. None of the biovar 1A strains was positive for the low calcium response reaction, which is known to be plasmid-encoded in pathogenic strains.
Size of plasmids of biovar lA strains In order to determine the size of some representative plasmids, restriction enzyme analysis was performed and the molecular weight of the plasmids was calculated on the basis of the size of the restriction fragments generated. For plasmid size determination we selected representatives from different habitats (environment, food, clinical isolates) containing plasmids migrating above the chromosomal DNA and three strains containing a small plasmid migrating below the chromosomal DNA. The virulence plasmid of a pathogenic strain was used as a reference. The results are shown in Table 3. The small plasmids had sizes between 2.7 and 6.3 kb. The large plasmids were of sizes ranging between about 50 and 70 kb.
Restriction patterns of plasmids of biovar lA strains The existence of large plasmids in biovar 1A strains indicated that there might be some similarity between these plasmids and the virulence plasmid. To answer this question, we compared the restriction patterns of a virulence plasmid with the patterns of several large plasmids of biovar 1A strains. The strains were chosen as representatives of the different habitats used for strain isolation (environmental isolate from a stream, food isolate, clinical isolate). Fig. 2 shows the patterns for the restriciton enzymes EcoRI, HindIII, and BamHI. The restriction patterns of the plasmids of the biovar 1A strains were different from the pattern of the virulence plasmid for all enzymes tested. These results do not point to a high degree of structural relationship between the virulence plasmid and the selected plasmids in biovar 1A strains.
Homology between the virulence plasmid and plasmids of biovar lA strains To obtain more information concerning possible similarities between the respective DNA sequences, we performed Southern blots with consecutive hybridization between the plasmids of the biovar 1A strains and the virulence plasmid of strain #13169. Table 3. Sizes of selected plasmids of Y. enterocolitica biovar 1 A strains compared with a virulence plasmid (p13169) strain #
estimated plasmid size (kb)
#13169
75
#78 #15673 #29845
52 65 70
#519 #29807 #29881
4.3 2.7 6.3
Plasmids of Y. enterocolitica biovar 1A
59
kb 23.1 9.4 6.6
4.4
2.3 2.0
0.6
EcoRI
HindlH
BamHI
Fig. 2. Comparison of restriction patterns of plasmids of Y. enterocolitica biovar 1A (#78, #15673, #29845) to a virulence plasmid (#13169).
The plasmids of 16 strains of Y. enterocolitica biovar 1A were digested with restriction enzymes, the fragments were separated by gel electrophoresis, transferred to a nylon membrane and hybridized with the labelled virulence plasmid of strain #13169. While the plasmids from 15 out of the 16 strains did not hybridize, the plasmid of strain #15673 showed some homology to the virulence plasmid indicating a corresponding similarity between the plasmid of the biovar 1A strain #15673 and the virulence plasmid of strain #13169. Fig. 3 shows the homology between the plasmid of strain #15673 and the virulence plasmid of strain #13169. Discussion
Y. enterocolitica biovar 1A strains, which are ubiquitously distributed and can be found in soil, water and food, are usually considered to be apathogenic. They can, however, sometimes be found in clinical isolates. This observation - together with the observation that biovar 1A strains sometimes contain large plasmids - raises the question as to whether the epidemiological importance of these strains may be underestimated. To answer this question, we analysed a collection of biovar 1A strains for their plasmid content and their in-vitro virulence-associated properties. Furthermore, the plasmids were examined for a possible relationship to a typical virulence plasmid of a pathogenic Y. enterocolitica strain.
60
A. Lewin et al.
Fig. 3. Homology between the virulence plasmid of Y. enterocolitica #13169 to the virulence plasmid of Y. pseudotuberculosis #29827 and to the plasmid of the Y. enterocolitica biovar 1A strain #15673. The plasmids were digested with the restriction enzyme HindIII and the fragments were separated in an agarose gel (A). The DNA was transferred from the gel to a membrane and hybridized with the fluorescein-labelled virulence plasmid of strain #13169 (B).
In a first step, biotyping was performed by routine diagnostic methods. 42 strains which could be classified as biovar 1A by these reactions were selected for analysis of their plasmid content. Our findings are in agreement with the data of other authors (e. g. 10,14,15) showing that plasmids are widely distributed in Yersinia strains and are not restricted to pathogenic strains. 38% of the selected biovar 1A strains carried plasmids most of which migrated to about the same position in an agarose gel as did the virulence plasmid under standard conditions. In routine diagnostic laboratories, these plasmids might thus be classified as virulence plasmids. The size of selected large plasmids was determined to be between 50 and 70 kb, whereas the size of the selected small plasmids was estimated to be between 2.7kb and 6.3kb.
Plasmids of Y. enterocolitica biovar 1A
61
Since these results raise the question whether in particular the large plasmids might carry some pathogenicity factors, we tested the strains for in-vitro virulence-associated characteristics. None of the strains classified as biovar lA was positive for the low calcium response reaction. However, many of the strains indeed possess some other virulence-associated characteristics as shown in Table 2. This result can be interpreted in two ways: Either the biovar lA strains have some virulence-associated characteristics and thus some pathogenic potential, or the in-vitro tests usually performed for the identification of pathogenic strains are not suitable for the biovar lA strains. The fact that biovar lA strains can also be isolated in connection with pathological events (16) supports the first hypothesis. On the other hand, the possibility that the tests for virulence-associated properties usually performed do not allow a reliable assessment of the virulence of biovar lA strains is in agreement with the observations of other authors (11). Both possibilities point to the necessity of applying methods different from the commonly used biochemical tests in order to determine the pathogenicity of Yersinia strains. Furthermore, the relevance of the existence of large plasmids in these strains should be investigated. These conclusions are in agreement with the results of Wachsmuth et al. (1984), who found that routine or simple plasmid profile analysis has a limited application in the diagnosis and epidemiology of infections caused by yersiniae. For these reasons, we applied molecular techniques for a further characterisation of the plasmids detected in our work. A comparison of the restriction patterns of selected large plasmids with the restriction pattern of a virulence plasmid showed that there were no similarities on this level. Work is in progress to analyse the plasmids of biovar lA strains to find out whether these plasm ids are related to each other or can be divided into subgroups. In most cases our hybridizations of the complete virulence plasmid to plasmids of biovar lA strains detected no homology at all. Interestingly, one strain (#15673) showed some homology to the virulence plasmid. Plasmid p15673 has a size of about 65 kb and thus a size similar to that of the virulence plasmids of pathogenic Yersinia strains. The restriction pattern of p15673, however, does not resemble the restriction pattern of the virulence plasmid. We are now performing further hybridizations to determine exactly which regions of the virulence plasmid are homologous to the plasmid of strain #15673. Preliminary results have localized the homologous region to sequences of the virulence plasmid which are outside the conserved calcium-regulation region (data not shown). It is noteworthy that strain #15673 is a clinical isolate (from the abomasum from an aborted calf foetus) and in addition has some in-vitro virulence-associated characteristics. This may indicate that the determination of a possible virulence of strains able to persist within organs requires virulence tests which are different from those commonly used. The homology between the virulence plasmid and the plasmid of biovar lA strain #15673 raises the question as to whether the homologous regions carry any virulence-associated genes, and whether these regions provide any potential for pathogenicity in such biovar lA strains. Our future work will concentrate on the analysis of the homologous sequences of p15673 and of the virulence plasmid in order to determine their biological function and their clinical importance. Acknowledgements. We thank B. Karbowiak and S. Wolgast-Hoffmann for excellent technical assistance. We also thank Dr. Feuerpfeil, Dr. Stephan, and D. Knabner for isolating and providing bacterial strains.
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A. Lewin et al. References
1. Aleksic, S. and J. Bockemuhl: Proposed revision of the Wauters et al. antigenic scheme for serotyping of Yersinia enterocolitica. J. Clin. Microbiol. 20 (1984) 843-845 2. Aleksic, S. and J. Bockemuhl: Mikrobiologie und Epidemiologie der Yersiniosen. Immun.Infekt. 18 (1990) 178-185 3. Aulisio, C. C. G., W. E. Hill, J. T. Stanfield, and R. L. jr. Sellers: Evaluation of virulence factor testing and characteristics of pathogenicity in Yersinia enterocolitica. Infect. Immunol. 40 (1983) 330-335 4. Birnboim, H. A rapid alkaline extraction method for the isolation of plasmid DNA. Meth. Enzymol. 100 (1983) 243-255 5. Birnboim, H. C. andJ. Doly: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7 (1979) 1513-1522 6. Heesemann, J.: Enteropathogene Yersinien: Pathogenitatsfaktoren und neue diagnostische Methoden. Immun. Infekt. 18 (1990) 186-191 7. Heesemann, J. and R. Laufs: Construction of a mobilizable Yersinia enterocolitica virulence plasmid. J. Bacteriol. 155 (1983) 761-767 8. Kado, C. I. and S. T. Liu: Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol. 145 (1981) 1365-1373 9. Karib, H. und H. Seeger: Vorkommen von Yersinien- und Campylobacter-Arten in Lebensmitteln. Fleischwirtsch. 74 (1994) 1104-1106 10. Kleemann, J.: Untersuchungen zum Vorkommen und Verhalten von Yersinia enterocolitica in Rohwurst. Diss., Berlin, 1993 11. Kleemann, J. und T. Bergann: Yersinia spp. in frischer Rohwurst. Fleischwirtsch. 74 (1994) 1101-1103 12. Kuznetzov V. G., V. N. Bagriantsev, and V. M. Kitaev: Vydelenie shtammov Yersinia intermedia s plazmidoi molekuiarnoi massy 82 mega dalton v prirodnykh pohuliatsiiakh iersinii. (The isolation of Yersinia intermedia strains with a plasmid of 82-megadalton molecular weight in natural populations of Yersinia). Zhurnal Mikrobiologii, Epidemiologii i Immunobiologii 11 (1990) 12-16 13. Maniatis, T., E. F. Fritsch, and J. Sambrook: Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1982 14. Marranzano, M., A. Agodi, and M. Gulisano: Molecular characterization of Yersinia strains isolated from human and environmental sources. Microbiologica 16 (1993) 5762 15. Mingrone, M. G. and M. Fantasia: Characteristics of Yersinia spp. isolated from wild and zoo animals. J. Wildlife Dis. 24 (1988) 25-29 16. Nattermann, H.: Untersuchungen zur Diagnostik, Epizootiologie, Pathogenese und Klinik der Yersinia enterocolitica-Infektion bei Schweinen, Schafen, Hiihnern und Hunden. Vet.-Med. Diss. B, HU Berlin (1986) 17. Nattermann, H.: Diagnose von Yersinia enterocolitica. Mitteilung 111993 der 12. AVIDTagung am 24. und 25. Juni 1993 in Berlin, Anlage VIII S.l 18. Nattermann, H., F. Horsch, K. Maier und D. Beutling: Neue Erkenntnisse zur Yersinia enterocolitica-Infektion des Schweines. Deutsche Veterinarmedizinische Gesellschaft, Tagung der Fachgruppe "Bakteriologie und bakterielle Krankheiten" Schloss Rauischholzhausen bei Marburg, 8.-10 Juni 1994, Abstr. in: Tagungsband S. 251-259 19. Prpic, J. K., R. M. Robins-Browne, and R. B. Davey: In vitro assessment of virulence in Yersinia enterocolitica and related species. J. Clin. Microbiol. 22 (1985) 105-110 20. Tauxe, R. V., J. Vandepitte, G. Wauters, S. M. Martin, V. Goossens, P. De Mol, R. van Noyer, and G. Thiers: Yersinia enterocolitica infections and pork: the missing link. Lancet 1 (1987) 1129-1132 21. Wachsmuth, K., B. A. Kay, and K. A. Birkness: Diagnostic value of plasmid analyses and assays for virulence in Yersinia enterocolitica. Diagn. Microbiol. Infect. Dis. 2 (1984) 219-228
c.:
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22. Wauters, G.: Carriage of Yersinia enterocolitica serotype 3 by pigs as a source of human infection. Contr. Microbiol. Immunol. 5 (1979) 249-252
23. Wauters, G., K. Kandala, and M. Janssens: Revised biogrouping scheme of Yersinia enterocolitica. Cont. Microbiol. Immunol. 9 (1987) 14-21 Prof. Dr. Bernd Appel, Robert Koch-Institut, Fachbereich 5, Nordufer 20, D-13353 Berlin, Germany