INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Oct. 1988, p. 424-429 Vol. 38, No. 4 0020-7713/88/040424-06$02.OO/O Copyright 0 1988, International Union of Microbiological Societies Yersinia mollaretii sp. nov. and Yersinia bercovieri sp. nov., Formerly Called Yersinia enterocolitica Biogroups 3A and 3B GEORGES WAUTERS,l* MICHELE JANSSENS,' ARNOLD G. STEIGERWALT,2 AND DON J. BRENNER2 Microbiology Unit, Faculty of Medicine, Catholic University of Louvain, Brussels, Belgium, and Molecular Biology Laboratory, Meningitis and Special Pathogens Branch, Division of Bacterial Diseases, Centers for Disease Control, Atlanta, Georgia 303332 Yersiniu enterocolitica biogroups 3A and 3B are biochemically, serologically, and ecologically different from biogroup 3 and other Y. enterocolitica biogroups. Both biogroup 3A and biogroup 3B can be characterized by their negative Voges-Proskauer reactions and positive reactions in tests for pyrazinamidase, acid production from mucate, proline peptidase, and acid production from D-xylose. Biogroup 3A ferments L-sorbose but not L-fucose; biogroup 3B has the opposite fermentation pattern. Deoxyribonucleic acid relatedness studies (hydroxyapatite method) indicated that biogroups 3A and 3B are two new species that are about 55% interrelated and 25 to 46% related to other Yersiniu species (except Yersinia ruckeri [20 to 22%]). The names Yersiniu mollaretii sp. nov. and Yersinia bercovieri sp. nov. are proposed for biogroups 3A and 3B, respectively. In addition to Yersinia enterocolitica sensu strict0 (3, Virulence markers. Autoagglutination after growth in several related species, previously known as Y. enteroco- broth at 37°C was tested in tryptic soy broth (17), and litica-like organisms, have been described. These are Yersi- calcium dependency was tested at 37°C on magnesium- nia intermedia (lo), Yersinia frederiksenii (19), Yersinia oxalate agar (14). kristensenii (8), Yersinia aldovae (7), and, most recently, Serotyping. 0 antigens were determined by the slide Yersinia rohdei (2). In 1978, Bercovier et al. described agglutination test, using 57 antisera against Y. enterocolitica strains originating from a terrestrial ecosystem and resem- and related Yersinia species (20). In addition, antisera were bling Y. enterocolitica biogroup 3 (4). The strains which raised against three new 0 factors designated 0:58, 059, fermented L-sorbose and either i- or myo-inositol were and 0:62. designated biogroup 3A; the strains which did not ferment DNA studies. Cells were grown at 37°C with shaking to these carbohydrates were designated biogroup 3B. In 1986, stationary phase in 1.5 liters of brain heart infusion broth Kaneko and Maruyama (16) isolated human pathogenic contained in 2-liter Erlenmeyer flasks. The methods used strains of serotype 3 in Japan that were Voges-Proskauer- for DNA isolation and purification, for DNA hybridization negative variants of biogroup 3. Typical serotype 3 strains by the hydroxyapatite method, and for guanine-plus-cy- are Voges-Proskauer positive and belong to biogroup 4. The tosine (G+C) content determinations have been described Voges-Proskauer-negative serogroup 3 strains were referred previously (11, 18). DNA was labeled in vitro with [32P] to as biogroup 3B by Fukushima (13). Wauters et al. (21) deoxyribose cytidine triphosphate by using a nick translation proposed that biogroup 3A and 3B strains be transferred to a reagent kit (Bethesda Research Laboratories, Inc., Gaithers- newly designated biogroup 6, to avoid confusion with bio- burg, Md.) and following the instructions of the manufac- group 3. turer. Differences in the ecology and the 0 and H antigenic patterns of biogroups 3A and 3B compared with biogroup 3 RESULTS prompted further biochemical and deoxyribonucleic acid DNA hybridization. Levels of DNA relatedness were (DNA) hybridization investigations of these strains. The determined by using labeled DNA from Y. mollaretii CNY results of these studies indicate that biogroups 3A and 3B are 7263T (T = type strain) and Y. bercovieri CNY 7506T (Table two new Yersinia species, for which the names Yersinia 2). All biogroup 3A strains tested were 74% or more related mollaretii (biogroup 3A) and Yersinia bercovieri (biogroup to strain CNY 7263T in both 60 and 75°C reactions (average, 3B) are proposed. 88% at 60"C, 89% at 75°C); the amount of divergence in related sequences was 0.5 to 2.5%. Similarly, with one MATERIALS AND METHODS exception, all biogroup 3B strains were at least 79% related to strain CNY 7506T in both 60 and 75°C reactions (average, Bacterial strains. The biogroup 3A and 3B strains which 86% at 60"C, 92% at 75°C); the amount of divergence in we used are listed in Table 1. The strains of other biogroups related sequences was 0.5%. The exception, biogroup 3B and other Yersinia species used in DNA hybridization stud- strain WE 120/83, was 56% related to Y. bercovieri at 60°C ies have been described previously (2,5, 7,8,19). All strains with 6.0% divergence and 41% related at 75°C; it was 39% were grown and maintained at room temperature on tryptic related to Y. mollaretii at 60°C with 9.0% divergence. The soy agar. levels of relatedness between Y. mollaretii and Y. bercovieri Biochemical properties. Biochemical tests were done by strains were 51 to 62% at 60°C with 5.0 to 7.5% divergence using the methods of Ewing (12) and Bercovier et al. (3). and 38 to 47% at 75°C. Y. mollaretii and Y. bercovieri were Pyrazinamidase activity (15) and proline peptidase (21) were 25 to 46% related to other Yersinia species (except Y. ruckeri tested as previously described. All tests were done at 25°C [20 to 22%]). Their levels of relatedness to Y. enterocolitica, and read at 1, 2, and 7 days, unless otherwise stated. including both Voges-Proskauer-positive and Voges-Pros- kauer-negative strains of biogroup 3, were no higher than * Corresponding author their levels of relatedness to most other Yersinia species. 424 VOL. 38, 1988 Y. MOLLARETII AND Y. BERCOVIERI 425 TABLE 1. Origins and serological characteristics of the Y. mollaretii and Y. bercovieri strains studied Strain" Source Country Received from: 0 antigensb Y. mollaretii WAIP 204T (= CDC Soil United States Bercovier 0:59,(20,36,7) 2465-87T = CNY 7263= = ATCC 43969T) WAIP 205 (= CNY 7264) Soil United States Bercovier 0:59,(20,36,7) WAIP 206 (= CNY 7442) Human stool The Netherlands Bercovier 0:59,(20,36,19) WA 291 Human stool Federal Republic of Germany Aleksic (H87-36/82) 0:3 WA 309 Raw vegetables France Catteau 0:59,(20,36,7) WA 739 Human stool The Netherlands Banffer 0:59,(20,36,19) WA749 Drinking water Federal Republic of Germany Ale ksic (H241-36/84) 0:6,30 WA 754 Human stool Finland Skurnik (10 Ye 92/84) 0: 59, (20,36,7) WA 751 Drinking water Federal Republic of Germany Aleksic (H253-36/84) 0:59,(20,36,19) WAT 932 Unknown Canada Toma (T9120) 0 :59, (20,36,7,19) WA942 Drinking water Federal Republic of Germany Aleksic (H412-36/83) 0:6,30 WA 989 Meat Austria Breuer 0:62,22 WE 316184 Human stool Belgium Nagels NT WS 19/86 Human stool Belgium Vandepitte 0:62,22 WS 20186 Human stool Belgium Van Noyen 0:7,13 WS 35/87 Human stool Belgium Van Noyen 0:62,22 W 787180 Human stool Belgium Wau ters 0:59,(20,36,7,19) Y. bercovieri WAIP 20gT (= CDC Human stool France Bercovier 0:58,16 2475-87T = CNY 7506' - ATCC 43970*) WAIP 207 (= CNY 7393) Human stool France Bercovier 0:58,16 WAIP 209 (= CNY 7966) Soil France Bercovier 0:18 WE 120183 Human stool Belgium Vandepitte 0:58,16 WA 313 Raw vegetables France Catteau 0:8 WA 314 Raw vegetables France Catteau 0:8 WA 315 Raw vegetables France Catteau 0:10 WA 317 Raw vegetables France Catteau Autoagglutinable WA 318 Raw vegetables France Catteau 0:5 WA 755 Human stool Finland Skurnik (12 Ye 127184) NT WA 760 Human stool Finland Skurnik (53 Ye 3016184) 0:58,16 WA 43/87 River water Norway Nesbakken 0:58,16 WE 187/80 Human stool Belgium Vandepitte 0:58,16 WE 580185 Human stool Belgium Wautele t 0:58,16 WE 171186 Human stool Belgium Collard 0:58,16 WE 83/86 Human stool Belgium Serruys 0:58,16 WE 188/86 Human stool Belgium Pepersack NT WE 68/87 Human stool Belgium Van Noyen 0:58,16 WA 763 Human stool Finland Skurnik 0:58,16 WS 235 Raw vegetables Belgium Wauters 0:58,16 W 601 Human stool Belgium Wauters 0:8 W 991 Human stool Belgium Wauters 0:58,16 WA 114 (= CNY 129) COYPU Great Britain Mair 0:8 a Abbreviations: W, WAIP, WA, WAT, WE, and WS, Laboratory in Brussels, Belgium; CDC, Centers for Disease Control, Atlanta; CNY, Centre National des Yersinia, Institut Pasteur, Paris, France; ATCC, American Type Culture Collection, Rockville, Md. The antigens in parentheses are weak or irregular. NT, Nontypable. G+C contents. DNAs from Y. mollaretii CNY 7263T, Virulence markers. Tests for autoagglutination and cal- WAT 932, and WA 739 had G+C contents of 50,50, and 51 cium dependency at 37"C, indicating the presence of the mol%, respectively. DNAs from Y. bercovieri CNY 7506T, virulence plasmid in Y. enterocolitica, were negative for all WA 315, and WE 166/84 had G+C contents of 50 mol%. Y. mollaretii and Y. bercovieri strains tested. Assays were done in triplicate. Antigenic patterns. The 0 antigens found in both species Phenotypic characterization. Those characteristics com- are listed in Table 1. Some strains shared common 0 mon to all Y. mollaretii strains or all Y. bercovieri strains are antigens with Y. enterocolitica, especially 0:6,30 and 0:3 in given below in the species descriptions.