Molecular typing of Yersinia pseudotuberculosis by using an IS200 like element

0095-1137/96/$04.00

₁

0

Copyright

q

1996, American Society for Microbiology

Molecular Typing of Yersinia pseudotuberculosis by Using an

IS200-Like Element

MONIQUE ODAERT, PATRICK BERCHE,

AND

MICHEL SIMONET*

Institut National de la Sante´ et de la Recherche Me´dicale U411, Faculte´ de Me´decine Necker,

75730 Paris Cedex 15, France

Received 18 March 1996/Returned for modification 1 May 1996/Accepted 12 June 1996

The IS200-like insertion sequence (IS) is a 708-bp element recently found in Yersinia pestis. Its nucleotide

sequence is 85% identical to that of IS200 recovered in most Salmonella enterica isolates. It is also present in

multiple copies in Y. pseudotuberculosis. In contrast, this IS is found in some (biotype 1B strains) but not other

Y. enterocolitica strains and is absent in the nonpathogenic yersiniae: Y. frederiksenii, Y. kristensenii, Y.

interme-dia, Y. bercovieri, and Y. mollaretii. The number and locations of the ISs in the Y. pseudotuberculosis genome vary

among strains, resulting in a high degree of polymorphism, but IS fingerprints are stable after multiple

subcultures of clinical isolates. The discriminative power of IS typing is better than that of ribotyping and

almost as good as that of the time-consuming method of pulsotyping. Overall, IS200-like is a useful molecular

marker in determining the epidemiology of Y. pseudotuberculosis infections.

The genus Yersinia includes three pathogenic species: Y.

pestis (the causative agent of bubonic plague), Y. enterocolitica,

and Y. pseudotuberculosis, the last two being responsible mainly

for enteritis and mesenteric lymphadenitis, which are

some-times complicated with septicemia. Seven environmental,

non-pathogenic species, Y. frederiksenii, Y. kristensenii, Y.

interme-dia, Y. bercovieri, Y. mollaretii, Y. aldovae, and Y. rohdei, have

been described (37). Y. pestis causes epidemic or pandemic

infections, while Y. pseudotuberculosis and Y. enterocolitica are

mostly involved in sporadic infections. However, several

out-breaks involving the two enteropathogenic species, Y.

entero-colitica (especially in North America) and Y. pseudotuberculosis

(mostly in Japan), have been reported (3, 15, 21, 27–29, 32–35).

In addition to serotyping, biotyping, and phage typing, a variety

of DNA-based methods have been developed for Yersinia

typ-ing, including analysis of restriction fragment polymorphism in

chromosomal DNA or in the plasmid pYV (1, 4, 10, 12, 13, 17,

22, 24), a 70-kb plasmid harbored by all pathogenic strains and

responsible for bacterial virulence (for a review, see reference

5).

We recently discovered a novel insertion sequence (IS) in Y.

pestis (31). This 708-bp IS, integrated within the inv gene from

Y. pestis, has 85% nucleotide identity with IS200, an element

which was first described in Salmonella enterica (14). This IS,

designated the IS200-like element, was found in multiple

cop-ies (at least 15 to 20) within the genomes of several Y. pestis

strains. Y. pseudotuberculosis DNA hybridizes with a probe

internal to the IS200-like element, but the insertion element

was present in fewer copies than in Y. pestis in the limited

number of strains tested (31). The degree of similarity between

the Y. pestis and Y. pseudotuberculosis IS200-like elements is

unknown, but restriction fragment length polymorphism

re-sults with the PCR products of the IS elements from both

species were identical (unpublished results).

ISs can be used as molecular tools for typing pathogenic

microorganisms, including Salmonella enterica, Staphylococcus

aureus, and Mycobacterium tuberculosis (2, 19, 20, 23, 36). This

conclusion appears to be paradoxical at first sight because ISs

are mobile genetic elements; however, transposition events

occur at a very low frequency. IS100 and IS285 have been

identified in pathogenic yersiniae (9, 25, 26), but these

inser-tion elements have not previously been used as molecular

markers in epidemiological analyses of plague and other forms

of yersiniosis. The aim of this work was to study the usefulness

of IS200-like elements for discriminating among strains

asso-ciated with known serotypes of enteropathogenic yersiniae. IS

fingerprinting was performed with 20 epidemiologically

unre-lated strains of Y. pseudotuberculosis, and the results allowed

good discrimination of the strains. In contrast, this molecular

typing method is not useful for epidemiological studies of Y.

enterocolitica infections.

MATERIALS AND METHODS

Bacterial strains and growth conditions.The Yersinia strains used in this study

are listed in Table 1. They were obtained from the Centre National de Re´fe´rence des Yersinia, Institut Pasteur (Paris, France). Plasmid pYV was detected in bacteria by colony-blot hybridization with a probe consisting of a 5.3-kb BamHI fragment from the Ca21_{dependence locus of virulence plasmid pIB1 as}

previ-ously described (30). Bacteria were grown at 308C with aeration in Luria-Bertani broth or on agar.

Preparation of DNA probes.The IS200-like gene probe (421 bp) was obtained

by PCR amplification of pYV-cured Y. pestis 6/69Mc with the primers 59-TTCT TGTATCCTGGCCGT-39and 59-TGCGGTCTGGCAACT-39as previously de-scribed (31). The ribosomal 16S RNA (rrn) probe (about 1.5 kb) was obtained by PCR amplification of Y. pseudotuberculosis with the universal primer pair 59-G GTTACCTTGTTACGACTT-39and 59-AAGAGTTTGATCATGGCTCAG-39. Probes were labeled by random priming (Rediprime kit; Amersham France, Courtaboeuf, France) with [a-32_{P]dCTP (Amersham), and labeled DNA was}

separated from unincorporated nucleotides by Sephadex G-50 column (Pharma-cia, Uppsala, Sweden) chromatography.

Southern hybridization.DNA extracted from bacterial cells as previously

described (18) was digested with appropriate restriction endonucleases according to the manufacturer’s instructions (New England Biolabs, Beverly, Mass.). Re-stricted DNA fragments were separated by electrophoresis through a 0.8% agarose gel in Tris-borate or Tris-acetate buffer and then transferred to a nylon membrane (Boehringer Gmbh, Mannheim, Germany). Probes were hybridized at 65 or 688C in buffer containing 0.1% sodium dodecyl sulfate (SDS), 63SSC (13SSC is 0.15 M NaCl plus 0.015 M sodium citrate [pH 7.0]), and 0.05% nonfat milk. After hybridization, filters were washed twice at room temperature for 30 min in 23SSC and 0.1% SDS and then twice at 65 or 688C for 45 min in 0.23

SSC and 0.1% SDS. Nylon membranes were autoradiographed by exposure to X-OMAT film (Eastman Kodak, Rochester, N.Y.) at2808C.

Pulsed-field gel electrophoresis.Bacterial DNA was embedded in agarose

plugs as previously described (7). DNA fragments generated by restriction en-donuclease digestion were separated in a 1% agarose–Tris-borate buffer gel by

* Corresponding author. Mailing address: Laboratoire de

Bac-te

´riologie-Hygie

`ne, Faculte

´ de Me

´decine Henri Warembourg, 1 Place

de Verdun, 59045 Lille, France. Phone: (33) 20444597. Fax: (33)

20529361. Electronic mail address: [email protected].

2231

on May 15, 2020 by guest

http://jcm.asm.org/

electrophoresis with a CHEF apparatus (Bio-Rad, Paris, France) at 148C and at 6 V/cm with alternating pulses at a 1208angle in a 1- to 30-s pulse-time gradient for 40 h. The gels were stained with ethidium bromide. DNAs from bacterio-phage lambda concatemers were used as size markers.

RESULTS AND DISCUSSION

The distribution of IS200-like elements in the genomes of

the pathogenic yersiniae Y. pseudotuberculosis and Y.

enteroco-litica and in the nonpathogenic species Y. frederiksenii, Y.

kris-tensenii, Y. intermedia, Y. bercovieri, and Y. mollaretii was

in-vestigated by Southern blot analysis. Total bacterial DNA was

digested with HincII, a restriction endonuclease that does not

cut within the IS element from either Y. pestis or Y.

pseudotu-berculosis, and DNA fragments were hybridized with a 421-bp

probe internal to the IS200-like element.

[image:2.612.55.557.82.293.2]

Twenty strains of Y. pseudotuberculosis isolated from human

and animal infections in several different countries (Table 1)

were studied: 16 strains belonged to serotypes 1, 2, 3, 4, or 6,

and 4 were not typeable with specific antisera. All strains tested

contained the IS, and the number of copies and locations of the

FIG. 1. IS200-like element profiles of Y. pseudotuberculosis. Southern blot of HincII-digested DNA with an internal fragment of the IS200-like element labeled with [32_{P]dCTP. Lanes 1 to 5, serotype 1 strains; lanes 6 to 9, serotype 2 strains; lanes 10 to 12, serotype 3 strains; lanes 13 and 14, serotype 4 strains; lanes 15 and 16, serotype}

[image:2.612.154.460.442.693.2]

6 strains; lanes 17 to 20, untypeable strains. The positions and sizes (in kilobases) of HindIII fragments of bacteriophage lambda DNA are indicated on the left.

TABLE 1. Yersinia strains used in this study

Species Serotype Origin Country No. of strains

Y. pseudotuberculosis

1

Human, pig, and goat

France and Italy

5

2

Human and hare

France

4

3

Human and cow

Spain and Argentina

3

4

Human and sheep

United Kingdom

2

6

Guinea pig

Japan

2

Nontypeable

Human

France

4

Y. enterocolitica

American strains (biotype 1B)

8

Human

Norway and United States

2

13 and 18

Unknown

United States

1

13a and 13b

Unknown

United States

1

20

Unknown

United States

2

21

Unknown

United States

2

European strains (biotype non-1B)

3

Human

France and Belgium

4

9

Human

France and Belgium

3

Y. frederiksenii

Human

France

3

Y. kristensenii

Human and milk

France

3

Y. intermedia

Human and environment

France

3

Y. bercovieri

Human and environment

France

3

Y. mollaretii

Human and cow

France

3

on May 15, 2020 by guest

http://jcm.asm.org/

element in the genomes varied among strains, resulting in a

high degree of polymorphism (Fig. 1). Comparisons of the

IS200-like profiles of two parental strains harboring the

viru-lence plasmid pYV and their respective isogenic pYV-cured

derivatives indicated that most IS copies, if not all, were

in-serted into the bacterial chromosome (data not shown).

Eigh-teen different genomic DNA restriction patterns for the

IS200-like element were found among the 20 isolates. Some IS bands

were shared by several strains. Three of the four untypeable

strains (Fig. 1, lanes 18, 19, and 20) contained 13 copies of the

IS, with similar distributions occurring in the genomes. In the

fourth untypeable strain (Fig. 1, lane 17), there was one copy

in an

;

2.8-kb fragment instead of the 2.5-kb fragment

ob-served in strains 18, 19, and 20 (Fig. 1). By using restriction

endonuclease BamHI, EcoRI, or EcoRV, enzymes that do not

cut within the IS element, we were still unable to distinguish

the four untypeable strains (data not shown).

The stability of restriction patterns of Y. pseudotuberculosis

DNA for the IS200-like element was studied to assess the value

of this insertion element as a marker for typing. Five strains

containing 7 to 13 copies of the insertion element were

sub-cultured at 30

8

C on agar plates, and after 20 subcultures (i.e.,

about 600 generations), DNA of each strain was extracted and

digested with HincII and DNA fragments were hybridized with

the IS200-like probe. For all of the strains tested, we were

unable to detect any difference in IS patterns after these

mul-tiple passages (data not shown); the frequency of IS200-like

transposition is under investigation. Consequently, IS200-like

fingerprinting appears to be useful for typing Y.

pseudotuber-culosis.

We then compared the discriminative capacity of IS typing

with two other molecular typing methods, pulsotyping and

ribotyping. Y. pseudotuberculosis was typed by pulsed-field gel

electrophoresis of NotI-restricted DNA; NotI is a rare-cutting

enzyme (one site per 100 kb of Yersinia DNA) (16). This

method was previously reported to be very efficient for Y.

pseudotuberculosis typing (12). The resulting macrorestriction

patterns of the 20 Y. pseudotuberculosis strains each contained

about 20 fragments ranging in size from

;

40 to

;

300 kb (Fig.

2). The 20 strains could be classified into 19 pulsotypes

accord-ing to these electrophoretic patterns. Interestaccord-ingly, the four

untypeable strains that exhibited identical IS200-like profiles

displayed different macrorestriction patterns (Fig. 2, lanes 17,

18, 19, and 20). However, the two serotype 6 strains (Fig. 2,

lanes 15 and 16) had the same pulsotype but different IS types

(Fig. 1, lanes 15 and 16). Thus, the analytical performance of

IS typing was similar to that of pulsotyping, which is a

time-consuming method; however, the two methods were

comple-mentary since they allowed discrimination of all 20 strains used

in the study.

Finally, restriction fragment length polymorphism patterns

of the rDNA regions of Y. pseudotuberculosis were studied by

HindIII digestion and probing with the 16S rRNA probe. This

restriction endonuclease does not cut within the 1.5-kb probe

and was previously shown by Picard-Pasquier et al. (24) to

discriminate Y. pseudotuberculosis strains well (five ribotypes

were individualized among nine unrelated strains). Bands

con-taining the rrn gene were 7 to

.

12 kb (Fig. 3), and 14 ribotypes

were obtained among the 20 strains. One strain of serotype 1

(Fig. 3, lane 4) and one of serotype 2 (lane 7) exhibited the

[image:3.612.60.296.70.308.2]

FIG. 2. NotI restriction profiles of Y. pseudotuberculosis. DNA was digested with NotI, and fragments were separated by pulsed-field gel electrophoresis as described in Materials and Methods. Lanes 1 to 5, serotype 1 strains; lanes 6 to 9, serotype 2 strains; lanes 10 to 12, serotype 3 strains; lanes 13 and 14, serotype 4 strains; lanes 15 and 16, serotype 6 strains; lanes 17 to 20, untypeable strains. The positions and sizes (in kilobases) of bacteriophage lambda DNA concate-mers are indicated on the right.

FIG. 3. 16S rRNA profiles of Y. pseudotuberculosis. Southern blot of HindIII-digested DNA with a 16S rRNA probe labeled with [32_{P]dCTP. Lanes 1 to 5, serotype}

1 strains; lanes 6 to 9, serotype 2 strains; lanes 10 to 12, serotype 3 strains; lanes 13 and 14, serotype 4 strains; lanes 15 and 16, serotype 6 strains; lanes 17 to 20, untypeable strains. Size markers (1-kb DNA ladder) are indicated on the left.

on May 15, 2020 by guest

http://jcm.asm.org/

[image:3.612.119.495.558.697.2]

same 16S rRNA profile, and one strain of serotype 2 (lane 6)

and two strains of serotype 3 (lanes 11 and 12) similarly fell

into the same riboclass. Moreover, the two serotype 6 strains,

which were not discriminated by pulsotyping, displayed the

same rRNA pattern (lanes 15 and 16). Finally, the four

un-typeable strains (lanes 17 to 20) were separated into two

groups (lanes 17 and 20 and lanes 18 and 19) on the basis of

their 16S rRNA pattern. Consequently, the discriminatory

power of IS typing is better than that of ribotyping. In addition,

analysis of rRNA pattern is not easy because of the fact that

fragments containing the rrn genes were mostly of large size

and thus poorly separated by agarose gel electrophoresis.

We also looked for the presence of the IS in the other

enteropathogenic Yersinia sp., Y. enterocolitica. Several

bio-types of this species have been defined by Wauters, Kandolo,

and Janssens (38). Biotype 1B strains, mainly from North

America, are highly pathogenic and responsible for major

out-breaks (especially of serotype O:8), whereas strains belonging

to other biotypes are less pathogenic or not pathogenic. We

studied 15 strains, of which 8 were from biotype 1B (American

strains). Unlike for Y. pseudotuberculosis, the IS200-like probe

hybridized with only some of the Y. enterocolitica strains (Fig.

4): only four biotype 1B strains (serotypes O:8, O:13,18, and

O:13a,13b) gave strong hybridization signals (one or two

bands). However, several faint background bands were

de-tected for most of the American strains. These faint bands

were still detected when DNA-DNA hybridization was carried

out under higher stringency conditions (68

8

C); thus, the weak

intensities of most of the bands suggest the presence of another

repetitive element in biotype 1B strains, similar but not

iden-tical to the IS200-like element; cloning and sequencing of this

element from these strains is in progress. None of the seven

non-biotype 1B strains (four of serotype O:3 and three of

serotype O:9) hybridized with the IS200-like probe. These data

are an additional argument indicating that the two groups of Y.

enterocolitica correspond to two divergent clusters, as

previ-ously suggested by Caugant et al. (6). Production of

iron-repressible proteins and the siderophore yersiniabactin under

iron starvation conditions are characteristic traits of the highly

pathogenic biotype 1B strains (8, 11), and the IS200-like

ele-ment could be an additional marker of Y. enterocolitica

patho-genicity. This proposition is reinforced by the absence of this

element in the nonpathogenic Yersinia species Y. frederiksenii,

Y. kristensenii, Y. intermedia, Y. bercovieri, and Y. mollaretii

(three strains were tested for each species [data not shown]).

ACKNOWLEDGMENTS

We thank E. Carniel, V. Escuyer, and P. Trieu-Cuot for critical

readings of the manuscript, and E. Abachin for technical assistance.

This work was supported by INSERM and the Universite

´ Rene

´

Descartes (Paris V, France).

REFERENCES

1. Andersen, J. K., and N. A. Saunders. 1990. Epidemiological typing of

Yer-sinia enterocolitica by analysis of restriction fragment length polymorphisms

with a cloned ribosomal RNA gene. J. Med. Microbiol. 32:179–187. 2. Baquar, N., A. Burnens, and J. Stanley. 1994. Comparative evaluation of

molecular typing of strains from a national epidemic due to Salmonella

brandenburg by rRNA gene and IS200 probes and pulsed-field gel

electro-phoresis. J. Clin. Microbiol. 32:1876–1880.

3. Black, R. E., R. J. Jackson, T. Tsai, M. Medvesky, M. Shayegani, J. C. Feeley,

K. I. E. McLeod, and A. M. Wakelee.1978. Epidemic Yersinia enterocolitica

infection due to contaminated chocolate milk. N. Engl. J. Med. 298:76–79. 4. Blumberg, H. M., J. A. Kiehlbauch, and I. K. Wachsmuth. 1991. Molecular epidemiology of Yersinia enterocolitica O:3 infections: use of chromosomal DNA restriction fragment length polymorphisms of rRNA genes. J. Clin. Microbiol. 29:2368–2374.

5. Brubaker, R. R. 1991. Factors promoting acute and chronic diseases caused by yersiniae. Clin. Microbiol. Rev. 4:309–324.

6. Caugant, D. A., S. Aleksic, H. H. Mollaret, R. K. Selander, and G. Kapperud. 1989. Clonal diversity and relationships among strains of Yersinia

enteroco-litica. J. Clin. Microbiol. 27:2678–2683.

7. Che´ron, M., E. Abachin, E. Gue´rot, M. El-Bez, and M. Simonet. 1994. Investigation of hospital-acquired infections due to Alcaligenes denitrificans subsp. xylosoxydans by DNA restriction fragment length polymorphism. J. Clin. Microbiol. 32:1023–1026.

8. de Almeida, A. M. P., A. Guiyoule, I. Guilvout, I. Iteman, G. Baranton, and

E. Carniel.1993. Chromosomal irp2 gene in Yersinia: distribution,

expres-sion, deletion and impact on virulence. Microb. Pathog. 14:9–21. 9. Filippov, A. A., P. N. Oleinikov, V. L. Motin, O. A. Protsenko, and G. B.

Smirnov.1995. Sequencing of two Yersinia pestis IS elements, IS285 and

IS100. Contrib. Microbiol. Immunol. 13:306–309.

10. Fukushima, H., M. Gomyoda, S. Aleksic, and M. Tsubokura. 1993. Differ-entiation of Yersinia enterocolitica serotype O:5, 27 strains by phenotypic and molecular techniques. J. Clin. Microbiol. 31:1672–1674.

11. Heesemann, J. 1987. Chromosomal-encoded siderophores are required for mouse virulence of enteropathogenic Yersinia species. FEMS Microbiol. Lett. 48:229–233.

12. Iteman, I., H. Najdenski, and E. Carniel. 1995. High genomic polymorphism in Yersinia pseudotuberculosis. Contrib. Microbiol. Immunol. 13:106–111. 13. Kapperud, G., T. Nesbakken, and K. Dommarsnes. 1991. Application of

restriction endonuclease analysis and genetic probes in the epidemiology of

Yersinia enterocolitica infection. Contrib. Microbiol. Immunol. 12:68–74.

14. Lam, S., and J. R. Roth. 1983. IS200: a Salmonella-specific insertion se-quence. Cell 34:951–960.

15. Lee, L. A., A. R. Gerber, D. R. Lonsway, J. D. Smith, G. P. Carter, N. D.

Puhr, C. M. Parrish, R. K. Sikes, R. J. Finton, and R. V. Tauxe.1990.

Yersinia enterocolitica O:3 infections in infants and children, associated with

the household preparation of chitterlings. N. Engl. J. Med. 322:984–987. 16. Lucier, T. S., and R. R. Brubaker. 1992. Determination of genome size,

macrorestriction pattern polymorphism, and nonpigmentation-specific dele-tion in Yersinia pestis by pulsed-field gel electrophoresis. J. Bacteriol. 174: 2078–2086.

17. Makino, S.-I., Y. Okada, T. Maruyama, S. Kaneko, and C. Sasakawa. 1994. PCR-based random amplified polymorphic DNA fingerprinting of Yersinia

pseudotuberculosis and its practical applications. J. Clin. Microbiol. 32:65–69.

18. Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3:208–218.

19. Millemann, Y., M.-C. Lesage, E. Chaslus-Dancla, and J.-P. Lafont. 1995. Value of plasmid profiling, ribotyping, and detection of IS200 for tracing avian isolates of Salmonella typhimurium and S. enteritidis. J. Clin. Microbiol.

33:173–179.

[image:4.612.59.295.71.298.2]

20. Monzon-Moreno, C., S. Aubert, A. Morvan, and N. El Sohl. 1991. Usefulness of three probes in typing isolates of methicillin-resistant Staphylococcus FIG. 4. IS200-like profiles of Y. enterocolitica. Southern blot of

HincII-di-gested DNA with an internal fragment of the IS200-like element labeled with [32

P]dCTP. Lanes 1 to 8, biotype 1B strains; lanes 9 to 15, biotype non-1B strains. Size markers (1-kb DNA ladder) are indicated on the left.

on May 15, 2020 by guest

http://jcm.asm.org/

aureus (MRSA). J. Med. Microbiol. 35:80–88.

21. Morse, D. L., M. Shayegani, and R. J. Gallo. 1984. Epidemiologic investi-gation of a Yersinia camp outbreak linked to a food handler. Am. J. Public Health 74:589–592.

22. Najdenski, H., I. Iteman, and E. Carniel. 1994. Efficient subtyping of patho-genic Yersinia enterocolitica strains by pulsed-field gel electrophoresis. J. Clin. Microbiol. 32:2913–2920.

23. Pelkonen, S., E.-L. Romppanen, A. Siitonen, and J. Pelkonen. 1994. Differ-entiation of Salmonella serovar infantis isolates from human and animal sources by fingerprinting IS200 and 16S rrn loci. J. Clin. Microbiol. 32:2128– 2133.

24. Picard-Pasquier, N., B. Picard, S. Heeralal, R. Krishnamoorthy, and P.

Goullet.1990. Correlation between ribosomal DNA polymorphism and

elec-trophoretic enzyme polymorphism in Yersinia. J. Gen. Microbiol. 136:1655– 1666.

25. Podladchikova, O. N., G. G. Dikhanov, A. V. Rakin, and J. Heesemann. 1994. Nucleotide sequence and structural organization of Yersinia pestis insertion sequence IS100. FEMS Microbiol. Lett. 121:269–274.

26. Portnoy, D. A., and S. Falkow. 1981. Virulence-associated plasmids from

Yersinia enterocolitica and Yersinia pestis. J. Bacteriol. 148:877–883.

27. Randall, K. J., and N. S. Mair. 1962. Family outbreak of Pasteurella

pseudo-tuberculosis infection. Lancet i:1042–1043.

28. Sanbe, K., M. Uchimura, K. Koiwai, K. Takagi, H. Yazaki, Y. Nanayama,

and M. Ohtawara.1987. Community outbreak of Yersinia pseudotuberculosis

occurred among primary school children. J. Jpn. Assoc. Infect. Dis. 61:763– 771.

29. Sato, K., K. Ouchi, and M. Taki. 1983. Yersinia pseudotuberculosis infection in children, resembling Izumi fever and Kawasaki syndrome. Pediatr. Infect. Dis. 2:123–126.

30. Simonet, M., and S. Falkow. 1992. Invasin expression in Yersinia

pseudotu-berculosis. Infect. Immun. 60:4414–4417.

31. Simonet, M., B. Riot, N. Fortineau, and P. Berche. 1996. Invasin production by Yersinia pestis is abolished by insertion of an IS200-like element within the

inv gene. Infect. Immun. 64:375–379.

32. Tacket, C. O., J. Ballard, N. Harris, J. Allard, C. Nolan, T. Quan, and M. L.

Cohen.1985. An outbreak of Yersinia enterocolitica infections caused by

contaminated tofu (soybean curd). Am. J. Epidemiol. 121:705–711. 33. Tacket, C. O., J. P. Narain, R. Sattin, J. P. Lofgren, C. Konigsberg, Jr., R. C.

Rendtorff, A. Rausa, B. R. Davis, and M. L. Cohen.1984. A multistate

outbreak of infections caused by Yersinia enterocolitica transmitted by pas-teurized milk. JAMA 251:483–486.

34. Tertti, R., K. Granfors, O.-P. Lehtonen, J. Mertsola, A.-L. Ma¨kela¨, I. Va

¨li-ma¨ki, P. Ha¨nninen, and A. Toivanen.1984. An outbreak of Yersinia

pseudo-tuberculosis infection. J. Infect. Dis. 149:245–250.

35. Toyokawa, Y., Y. Ohtomo, and T. Akiyama. 1993. Large scale outbreak of

Yersinia pseudotuberculosis serotype 5a infection at Noheji-machi in Aomori

Prefecture. J. Jpn. Assoc. Infect. Dis. 67:36–43.

36. Van Soolingen, D., P. W. M. Hermans, P. E. W. de Haas, D. R. Soll, and

J. D. A. van Embden.1991. Occurrence and stability of insertion sequences

in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J. Clin. Microbiol. 29:2578–2586.

37. Wauters, G., M. Janssens, A. G. Steigerwalt, and D. J. Brenner. 1988.

Yersinia mollaretii sp. nov. and Yersinia bercovieri sp. nov., formerly called Yersinia enterocolitica biogroups 3A and 3B. Int. J. Syst. Bacteriol. 38:424–

429.

38. Wauters, G., K. Kandolo, and M. Janssens. 1987. Revised biogrouping scheme of Yersinia enterocolitica. Contrib. Microbiol. Immunol. 9:14–21.

on May 15, 2020 by guest

http://jcm.asm.org/