Kluyvera: Buttiamella Ferragutiae Sp

INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1996, p. 50-63 Vol. 46, No. 1 0020-7713/96/$04.00+0 Copyright 0 1996, International Union of Microbiological Societies

Emended Description of Buttiamella agrestis with Recognition of Six New Species of Buttiamella and Two New Species of Kluyvera: Buttiamella ferragutiae sp. nov., Buttiamella gaviniae sp. nov., Buttiamella brennerae sp. nov., Buttiamella izardii sp. nov., Buttiamella noackiae sp. nov., Buttiamella warmboldiae sp. nov., Kluyvera cochleae sp. nov., and Kluyvera georgiana sp. nov. HANS E. MULLER,'" DON J. BRENNER,2 G. RICHARD PATRICK A. D. GRIMONT,4 AND PETER UMPFER'? Staatliches Medizinaluntersuchungsamt, 0-38124 Braunschweig, and Fachgebiet Hygiene, Technische Universitat, D-13353 Berlin' Germany; Emerging Bacterial and Mycotic Diseases Branch, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 303332; Division of Biochemistry, Walter Reed Army Institute uf Research, Washington, D. C. 20307-51003; and Unit6 des Enterobacthies, Institut National de la Santk et de la Recherche Mddicale Unit&389, Institut Pasteur, F-75724 Paris Cedex 15, France4

A total of 219 strains belonging to the genera Buttiuuxellu and Kluyveru were studied; 171 of these strains were isolated from mollusks, mainly snails and slugs, obtained from around the world. On the basis of DNA-DNA hybridization data, the strains were grouped into 11 genomospecies. A total of 44 phenotypic characters were used to differentiate the genera Buttiuuxellu and Kluyveru at the genus level and to identify genomospecies. There were significantly higher phenotypic probability distances between the genomospecies in the genus Butti- uuxella and the genomospecies in the genus Kluyveru than between the genomospecies in the same genus. There- fore, the existence of Buttiuuxellu and Kluyveru as different genera was confirmed. The existence of new species necessitated broadening the definitions of both genera. In two cases, two Buttiuuxellu species could not be quantitatively differentiated biochemically, and several other pairs of species could be separated only by the results of one biochemical test. Nonetheless, combinations of several characteristics were used to differentiate all of the species with levels of certainty ranging from log 10.79 to log 57.77 (calculated as probability distances). The following new species are proposed Buttiuuxellaferragutiue (type strain, ATCC 51602 [DSM 9390]), Buttiuuxellu guviniue (type strain, ATCC 51604 [DSM 9393]), Buttiuuxella brennerue (type strain, ATCC 51605 [DSM 9396]), Buttiuuxella izardii (type strain, ATCC 51606 [DSM 9397]), Buttiuuxellu nouckiue (type strain, ATCC 51607 [DSM 9401]), Buttiuuxellu wumzboldiae (type strain, ATCC 51608 [DSM 9404]), Kluyveru cochleae (type strain, ATCC 51609 [DSM 9406]), and Kluyveru georgiunu (type strain, ATCC 51603 [DSM 94091).

In 1976, a taxon designated group F was described by Gavini culture collection were more than 100 strains that were placed et al. (15). The strains belonging to group F were originally in a group designated enteric group 8. In 1981, Farmer et al. isolated from humans, soil, and water in the course of a study redefined the genus Kluyvera to include enteric group 8 along on the genus Citrobacter, and they were lactose positive, as are the named species Kluyvera ascorbata and Kluyvera cryocrescens members of the genus Citrobacter. However, these organisms and a third unnamed species (11). The genera Buttiauxella and differed from Citrobacter strains in several biochemical char- Kluyvera are generally similar phenotypically, although they acteristics, as well as in their DNA-DNA-hybridization char- can be differentiated. The members of these genera have dif- acteristics and guanine-plus-cytosine (G+ C) ratios (13, 14). ferent G+C contents and exhibit low levels of DNA related- Hence, the 17 strains were considered members of a new genus ness (14). in the family Enterobacteriaceae. This genus was named Butti- The frequent and profuse occurrence of Buttiauxella and auxella and contained a single species Buttiauxella agrestis. Kluyvera strains in snails and slugs is an intriguing finding. Four strains subsequently studied by Ferragut et al. were Numerous strains belonging to these previously rarely seen placed in the genus Buttiauxella but were not B. agrestis strains genera have been isolated. However, the phenotypic charac- (13). At the Centers for Disease Control and Prevention, three teristics that separate these genera did not allow clear assign- of these strains were placed in a group that was given the ment of these isolates at the genus level. The biochemical vernacular name enteric group 63, and the fourth was placed in reactions of the previously described species and the new iso- enteric group 64 (10). lates suggested that most of the new isolates belong to new Also in the Centers for Disease Control and Prevention species. In this paper we describe the results of a taxonomic study of Buttiauxella and Kluyvera strains. MATERIALS AND METHODS * Corresponding author. t Present address: Institut fur Angewandte Mikrobiologie, Justus- Bacterial strains. The Buttiauxella and Kluyveru strains used in this study are Liebig Universitat Giessen, D-35390 Giessen, Germany. listed in Table 1. The field strains were isolated from snails and slugs and samples

50 VOL. 46, 1996 NEW BUTTUUXELLA AND KLuM/ERA SPECIES 51

TABLE 1. Strains investigated in this study Serial Group Strain Labora- no. tory(ies)" Origin Other designation(s)b

DNA group 1 (B. agrestis) 001' CDC 1176-81T (= CUETM 77-167T) 1, 4 Unpolluted soil, Franced ATCC 33320T 002 CUETM 77-157 4 Drinking water, France" 003 CUETM 78-3 4 Unpolluted soil, Franced 004 CUETM 78-5 4 Unpolluted soil, Franced 005 CUETM 78-8 4 Drinking water, Franced 006 CUETM 78-18 4 Unpolluted soil, Franced 007 CUETM 78-19 4 Drinking water, France" 008 CUETM 78-27 4 Drinking water, France" 009 CUETM 78-29 4 Drinking water, Franced ACTT 33994 010 CUETM 78-34 4 Unpolluted soil, France" ATCC 33995 01 1 CUETM 78-44 4 Drinking water, Franced 012' S3/3-162 3 Snail, Braunschweig, Germany DSM 9388 013" S3/3-203 3 Snail, Braunschweig, Germany 014 S3/4-204 3 Snail, Braunschweig, Germany 015 S3/4-556 3 Snail, Braunschweig, Germany 016 s3/4-775 3 Snail, Braunschweig, Germany 017' S3/4-776 3 Snail, Braunschweig, Germany 018' s3/4-779 3 Snail, Braunschweig, Germany 019 S314-871 3 Snail, Braunschweig, Germany 020 S3/4-1 098 3 Slug, Braunschweig, Germany 021' S3/5-201 3 Snail, Braunschweig, Germany 022' s315-757 3 Snail, Braunschweig, Germany 023" S316-333 3 Slug, Braunschweig, Germany DSM 9389 024" S3/7-896 3 Slug, Braunschweig, Germany 025' S3/7-897 3 Snail, Braunschweig, Germany 026' 5318-910 3 Slug, Salisbury, Great Britain 027 S3/9-488 3 Slug, Braunschweig, Germany 028 S3/9-602 3 Snail, Braunschweig, Germany 02Y S3/9-878 3 Snail, Braunschweig, Germany 03F S3/9-887 3 Slug, Braunschweig, Germany 031' S3110-769 3 Snail, Braunschweig, Germany 032' S3/11-533 3 Slug, Braunschweig, Germany 033' S3111-794 3 Snail, Braunschweig, Germany 034 S3/11-832 3 Snail, Braunschweig, Germany 035 S3111-859 3 Snail, Braunschweig, Germany 036 s3114-770 3 Snail, Braunschweig, Germany 037" s3114-1071 3 Slug, Braunschweig, Germany 038 S3/14- 1073 3 Slug, Braunschweig, Germany 039 S3/14-1080 3 Slug, Braunschweig, Germany 040 s3114-108 1 3 Slug, Braunschweig, Germany 04 1 S3/14-1082 3 Slug, Braunschweig, Germany 042' S3/14-1085 3 Slug, Braunschweig, Germany 043 s3114-1094 3 Slug, Braunschweig, Germany 044 S3/14-1096 3 Slug, Braunschweig, Germany 045 s3114-1099 3 Slug, Braunschweig, Germany 046 S3/14-1101 3 Slug, Braunschweig, Germany 047 S3114-1 127 3 Slug, Braunschweig, Germany 048 S3/14-1129 3 Slug, Braunschweig, Germany 049 s3114-1132 3 Slug, Braunschweig, Germany 050 S3/14-1146 3 Slug, Braunschweig, Germany 05 1 S3/14-1149 3 Slug, Braunschweig, Germany 052 S3/14-1166 3 Slug, Braunschweig, Germany 053' S3/13-692 3 Slug, Braunschweig, Germany DNA group 2 (enteric 05 4' CDC 1180-81T (= CUETM 78-31T) 1, 4 Unpolluted soil, France (13) ATCC 51602=, DSM 9390T group 63, B. ferragutiae) 055 CUETM 78-50 4 Drinking water, France (13) DSM 9391 056 CUETM 78-35 4 Unpolluted soil, France (13) 05 7 CUETM "H. alvei 125" 4 Unknown, France

DNA group 3 (enteric 058' CDC 1175-81 (= CUETM 77-159) 1, 4 Unpolluted soil, France (13) DSM 9392 group 64, B. gaviniae) 059 Slll-895 3 Slug, Braunschweig, Germany 060 S1/1-978 3 Snail, Braunschweig, Germany 061 Slll-981 3 Snail, Braunschweig, Germany 062" Sl/l-984T 3 Snail, Braunschweig, Germany ATCC 51604, DSM 9393 063" S1/1-986 3 Snail, Braunschweig, Germany 064 s111-987 3 Snail, Braunschweig, Germany 065 s111-988 3 Snail, Braunschweig, Germany 066 s111-993 3 Snail, Braunschweig, Germany

~ Continued on following page 52 MULLER ET AL. INT. J. SYST.BACTERIOL.

TABLE 1-Continued Labora- Serial Strain Origin Other designation(s)'' Group no. tory( ies)" 067 S1/1-996 3 Snail, Braunschweig, Germany 068 Sl/1-998 3 Snail, Braunschweig, Germany 069 S1/1-999 3 Snail, Braunschweig, Germany 070 s lil-1000 3 Snail, Braunschweig, Germany 071 S 1/1-1003 3 Snail, Braunschweig, Germany 072 S 1/1-1007 3 Snail, Braunschweig, Germany 073 Sl/l-1032 3 Snail, Braunschweig, Germany 074 Sl/l-1033 3 Snail, Braunschweig, Germany 075 Sl/l-1039 3 Snail, Braunschweig, Germany 076 Sl/l-1041 3 Snail, Braunschweig, Germany 077 Sl/l-1049 3 Snail, Braunschweig, Germany 078 Sl/l-1050 3 Snail, Braunschweig, Germany 079 Sl/l-105 1 3 Snail, Braunschweig, Germany 080 Sl/l-1116 3 Slug, Braunschweig, Germany 08 1 s1/1- 1118 3 Slug, Braunschweig, Germany 082 Sl/l-1161 3 Slug, Braunschweig, Germany 083 Sl/l-1162 3 Slug, Braunschweig, Germany 084 S1/2-992 3 Snail, Braunschweig, Germany 085' S1/2-994 3 Snail, Braunschweig, Germany 086 s1/2-1020 3 Snail, Braunschweig, Germany 087" su2-1111 3 Slug, Braunschweig, Germany 088' S 1/3-1052 3 Snail, Braunschweig, Germany 089' S 1/3-1200 3 Snail, Braunschweig, Germany 090' S1/4-695 3 Slug, Braunschweig, Germany 09 1 S1/5-778 3 Snail, Braunschweig, Germany 092 S1/5-791 3 Snail, Braunschweig, Germany 093' S1/5-857 3 Snail, Braunschweig, Germany 094' S 115-860 3 Snail, Braunschweig, Germany 095' S 1/7-674 3 Snail, Braunschweig, Germany 096' S1 /7- 1 143 3 Slug, Braunschweig, Germany 097 S1/9-505 3 Slug, Braunschweig, Germany 098 S 1/9412 3 Slug, Braunschweig, Germany 099' S 1/9414 3 Slug, Braunschweig, Germany 100' S 1/9418 3 Slug, Braunschweig, Germany 101 S1/9-1177 3 Slug, Braunschweig, Germany 102 S 1/lo-549 3 Snail, Braunschweig, Germany 103' s 1110-801 3 Snail, Braunschweig, Germany 104' S1/10-853 3 Snail, Braunschweig, Germany 105' S1/10-863 3 Snail, Braunschweig, Germany 106 s l/lO-1044 3 Snail, Braunschweig, Germany 107 S1/10-1179 3 Slug, Braunschweig, Germany 108' S1/11-577 3 Snail, Braunschweig, Germany 109 Sl/ll-630 3 Snail, Braunschweig, Germany 1lo' Sl/ll-707 3 Slug, Braunschweig, Germany 111 S1/11-787 3 Snail, Braunschweig, Germany 112 S1/11-893 3 Slug, Braunschweig, Germany 113' s1/12-1180 3 Slug, Braunschweig, Germany 114' S 1/14-564 3 Snail, Braunschweig, Germany 115' S 1/14-669 3 Snail, Braunschweig, Germany 116 S1114-873 3 Snail, Braunschweig, Germany 117 S1/14-875 3 Snail, Braunschweig, Germany 118 SL'1.5-773 3 Snail, Braunschweig, Germany 119' S 1/15-774 3 Snail, Braunschweig, Germany 120 SU1.5-786 3 Snail, Braunschweig, Germany 121' S1/15-828 3 Snail, Braunschweig, Germany 122 S1/1.5-845 3 Snail, Braunschweig, Germany 123' S1/16-12 3 Slug, Braunschweig, Germany 124' S1/17-7 3 Slug, Braunschweig, Germany 125 S1/17-702 3 Slug, Braunschweig, Germany 126 S1/17-705 3 Slug, Braunschweig, Germany 127 S1117-903 3 Slug, Salisbury, Great Britain 128 S1/17-906 3 Slug, Salisbury, Great Britain 129' S1/17-911 3 Slug, Salisbury, Great Britain DSM 9394 130" S1/18-662 3 Snail, Braunschweig, Germany 131 S1/18-665 3 Snail, Braunschweig, Germany 132' S1/18-784 3 Snail, Braunschweig, Germany 133 S1/19-388 3 Slug, Braunschweig, Germany 134' S1/19-661 3 Snail, Braunschweig, Germany Continued on following page VOL. 46, 1996 NEW BUTTUUXELLA AND KLUYKERA SPECIES 53

TABLE 1-Continued

Serial Strain Labora- Origin Other designation(s)' Group no. tory(ies)u 135 S 1119-664 3 Snail, Braunschweig, Germany 136' S1/19-760 3 Snail, Braunschweig, Germany 137 S1/19-800 3 Snail, Braunschweig, Germany 138 S1/19-86 1 3 Snail, Braunschweig, Germany 139' EG59-like strain K 2258 3 Snail, Kaernten, Austria 140' EG59-like strain K 2286 3 Snail, Kaernten, Austria 141' EG59-like strain K 2340 3 Snail, Kaernten, Austria 142' EG59-like strain SA 1191 3 Snail, Cape Town, South Africa 143' EG59-like strain Z 2132 3 Snail, Zurich, Switzerland DSM 9395

DNA group 4 (B. bren- 144' S 116-360 3 Slug, Braunschweig, Germany nerae) 145' S1/6-571T 3 Snail, Braunschweig, Germany ATCC 51605T DSM 9396'r 146' S1/6-706 3 Slug, Braunschweig, Germany DSM 9413 147' S1/8-1022 3 Snail, Braunschweig, Germany 148' S1/8-783 3 Snail, Braunschweig, Germany 144 S3/8-879 3 Snail, Braunschweig, Germany 15V S3112-695 3 Snail, Braunschweig, Germany

DNA group 5 (B. izardii) 15 1' S3/2- 161T 3 Snail, Braunschweig, Germany ATCC 51606T, DSM 9397T 152' s312-199 3 Snail, Braunschweig, Germany DSM 9398 153 s312-5 25 3 Slug, Braunschweig, Germany 154 s3/2-589 3 Snail, Braunschweig, Germany 155 s3/2-917 3 Earthworm, Salisbury, Great Britain 156 S3/2-1113 3 Slug, Braunschweig, Germany 157 S3/2- 1178 3 Slug, Braunschweig, Germany 158' S3/10-487 3 Slug, Braunschweig, Germany 159' S3/13-626 3 Snail, Braunschweig, Germany 160' EG59-like strain NSW 150 3 Snail, Sydney, Australia 161' EG59-like strain NSW 155 3 Snail, Sydney, Australia DSM 9399 162' EG59-like strain RA 144 3 Snail, Rabat, Morocco DSM 9400 163' EG59-like strain RA 151 3 Snail, Rabat, Morocco

DNA group 6 (enteric 164' EG59-like strain BR 489 3 Snail, Sao Paulo, Brazil group 59, B. noackiue) 165' EG59-like strain BR 539 3 Snail, Sao Paulo, Brazil 166' EG59-like strain BR 588 3 Snail, Sao Paulo, Brazil 167' EG59-like strain BR 607 3 Snail, Sao Paulo, Brazil 168' EG59-like strain BR 626 3 Snail, Sao Paulo, Brazil 169' EG59-like strain BR 637 3 Snail, Sao Paulo, Brazil 170' EG59-like strain NSWllT 3 Snail, Sydney, Australia ATCC 51607T, DSM 9401'r 171' EG59-like strain NSW87 3 Snail, Sydney, Australia 172' EG59-like strain NSW 120 3 Snail, Sydney, Australia 173' EG59-like strain NSW 245 3 Snail, Sydney, Australia 174' EG59-like strain NSW 297 3 Snail, Sydney, Australia 175' EG59-like strain NSW 336 3 Snail, Sydney, Australia 176' EG59-like strain NSW 402 3 Snail, Sydney, Australia 177' EG59-like strain RA 116 3 Snail, Rabat, Morocco DSM 9402 178' EG59-like strain SA 1313 3 Snail, Cape Town, South Africa 179' CDC 1650-80 (= EG59) 1 Human clinical specimed DSM 9403

DNA group 7 (B. warm- 180" EG59-like strain NSW 184 3 Snail, Sydney, Australia boldiae) 181' EG59-like strain NSW 253 3 Snail, Sydney, Australia 182' EG59-like strain NSW 326 3 Snail, Sydney, Australia ATCC 5 1608T, DSM 9404T 183' EG59-like strain NSW 393 3 Snail, Sydney, Australia 184' EG59-like strain SA 420 3 Snail, Cape Town, South Africa DSM 9405

DNA group 8 (K. cochleae) 185' S3/1-49T 3 Snail, Braunschweig, Germany ATCC 51609T, DSM 9406T 186' s3/1-913 3 Slug, Salisbury, Great Britain DSM 9407 187' S3/6-835 3 Snail, Braunschweig, Germany

DNA group 9 (enteric 188 CDCICUETM 4246-74 1, 4 Human clinical specimeX DSM 9408 group 36/37, Kluyveru 189' CDC 2891-7fjT 1 Human sputumR ATCC 51603T, DSM 9409T species 3, K. georgiana) 190 CDCICUETM 2065-76 1, 4 Human clinical specimerP 191 CDCICUETM 2774-70 1, 4 Human clinical specimerP 192 CDCICUETM 3 108-76 1, 4 Human clinical specimeX

DNA group 10 (K. ascor- 193' CDCICUETM 648/74T 1, 4 Human sputum, North Caroling ATCC 33433T bata) 194 CDC 4450-74 1 UnknowrP 195 CDC 4417-74 1 Unknowd 196 CDCICUETM 762-75 1, 4 UnknownR 197 CDC 3858-69 1 UnknownR Continued on following page 54 MULLER ET AL. INT. J. SYST. BACTERIOL.

TABLE l-Continued

Serial Labora- Group Strain Origin Other designation(s)" no. tory( ies)"

198 CDC 1220-73 1 Unknowng 199 CDC 972-78 1 UnknownR 200 CDC 2070-78 1 Unknowng 20 1 CUETM 1-79 4 Unknown 202 CDC 2064-78 1 Unknowng 203 CDC 2589-70 1 UnknownR 204 CDC 2567-61 1 Sewage ATCC 14236 205 CDC 2221-78 1 Human (?), New Yorkg ATCC 33434 DNA group 11 (K. clyo- 206 CUETM 9-79 4 Unknown crescens) 207 CUETM 20-74 4 Unknown 208 CUETM 14-79 4 Unknown 209 CUETM 3-79 4 Unknown 210 CDC/CUETM 1734-74 1, 4 Unknowng 21 1 CDC/CUETM 1396-73 1, 4 Unknowng 212 CUETM 2-79 4 Unknown 213 CDC 410-78 1 Unknowng 214' CDC 2065-7ST 1 Kitchen food, Persian GulF ATCC 33435T 215 CDC 2568-61 1 Unknown ATCC 14237 216 CDC 2569-61 1 Unknown ATCC 14238 217 BU 005 12810484 2 Unknown DSM 9410 218 BU 009 33728683 2 Unknown DSM 941 1 219 BU 010 14812T 2 Unknown DSM 9412 '' 1, Centers for Disease Control and Prevention, Atlanta, Ga., 2, Fachgebiet Hygiene, Technische Universitat, Berlin, Germany; 3, Staatliches Medizinaluntersu- chungsamt, Braunschweig, Germany; 4, Institut Pasteur, Paris, France. 'ATCC, American Type Culture Collection; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen. ' P-labeled strain. "See reference 13. ' Strain used in DNA-DNA hybridization experiments.

f See reference 10. See reference 11.

were obtained from around the world. The strains were identified in two steps. assessed visually daily. In the esculin, hydroxyquinoline, (3-glucuronide, trypto- The first step involved screening tests to identify organisms that were members phan, and histidine tests development of a color (black, orange to brown, and of the family Enterobacieriaceae (rod-shaped, gram-negative, oxidase-negative, dark red, respectively) was considered a positive reaction (5). At the Technische catalase-positive organisms that fermented D-glucose, reduced nitrate, and ex- Universitat Berlin carbon source utilization tests were performed in microtiter hibited unusual L-arginine dihydrolase, L-lysine decarboxylase, and L-ornithine plates (flat-bottom wells) by using filter-sterilized M70 minimal medium (18) decarboxylase activity patterns [i.e., strains that were L-arginine dihydrolase supplemented with 0.002% (wt/vol) yeast extract (Oxoid) and 0.002% (wt/vol) negative, L-lysine decarboxylase negative, and L-ornithine decarboxylase positive, bio-Lactysat (bio-MCrieux). Each filter-sterilized carbon source was added at a strains that were L-arginine decarboxylase positive, L-lysine decarboxylase nega- final concentration of 0.2% (wt/vol). For aromatic compounds, a concentration tive, and L-ornithine decarboxylase negative, and strains that were L-arginine of 0.05% (wt/vol) was used. Qualitative enzyme tests were performed in filter- decarboxylase positive, L-lysine decarboxylase negative, and L-ornithine decar- sterilized media (pH 7.2) containing 0.05 M Tris HCI buffer, 0.05% yeast extract boxylase positive]). After this step additional tests (Table 2) were performed. (Oxoid), and 0.05% bio-Lactysat (bio-Merieux). Filter-sterilized solutions of DNA relatedness. DNA-DNA hybridization experiments were performed with chromogenic substrates (p-nitrophenyl-linked compounds) were added at a final 95 strains. To obtain DNA, cells were grown in brain heart infusion broth at 36 concentration of 2 mM. For p-nitroanilides, a concentration of 1 mM was used. t 1°C with shaking until they reached the late logarithmic phase. The methods The preparations were incubated at 30"C, and growth was assessed visually daily used to extract and purify DNA and the batch hydroxyapatite method used for for 4 days by comparing the cultures with a control that did not contain substrate. DNA-DNA hybridization have been described previously (6). An incubation Calculation of phenotypic probability distances. The probability distance (pd) temperature of 60°C was used for optimal DNA reassociation, and an incubation for each pair of species was calculated in two steps by the using average per- temperature of 75°C was used for stringent DNA reassociation. Labeled DNAs centages (vl and v2) obtained from discriminating tests (tl to tn). First, the larger from strains 001,012,054,058,062, 143, 145, 149, 151, 161, 170, 179, 182, and 185 of the two average percentages for two taxa was divided by the smaller, giving a (serial numbers [Table 11) were prepared enzymatically in vitro with [32P]dCTP value of >1 (v2/vl,,). Because this calculation failed for values of 0 and 100%. by using a nick translation reagent kit (Gibco BRL, Gaithersburg, Md.) as these values were replaced by 0.001 and 99,99%, respectively, without any prac- directed by the manufacturer. Levels of relatedness were expressed as relative tical disadvantage. Thereafter, n quotients of n tests were multiplied (pd = binding ratios, and the level of divergence of related sequences was determined v2/vl,, X v2/vlt,. . .X. . .v2/vlt,). Low values represented close probability dis- by calculating the decrease in thermal stability in a heteroduplex (reassociated tances and high levels of phenotypic relatedness and vice versa (Tables 3 through labeled and unlabeled DNAs from two different strains) compared with the 5). thermal stability in a homoduplex (reassociated labeled and unlabeled DNAs from the same strain). Levels of divergence were calculated to the nearest 0.5% on the assumption that each 1°C decrease in heteroduplex DNA stability was due RESULTS AND DISCUSSION to approximately 1% unpaired (diverged) bases within the related DNA (3,4, 11, 16). A species was defined as a group of strains that exhibited levels of DNA Habitat. The few previously described Buttiamella strains relatedness at the optimal reassociation temperature of 70% or more and whose were isolated from unpolluted soil and drinking water, surface related sequences exhibited 5% or less divergence (19). Biochemical tests. Routine biochemical tests were performed by using stan- water, sewage, soil, and fecal samples, and to our knowledge dardized procedures, and results were recorded after 48 h of incubation at 30 -+ no Buttiamella strains have been isolated from primary sterile 1°C (8, 9, 12). Consistent results for the decarboxylase reactions were obtained clinical specimens. Kluyvera strains have been isolated more by using the medium of Fay and Barry at pH 5.5 (1). Carbon source utilization frequently than Buttiamella strains but often appear to be tests were performed at the Institut Pasteur Paris by using Biotype strips (bio- MCrieux, La Balme les Grottes, France) that contained 99 pure carbon sources. biochemically different from members of the previously de- The strips were inoculated by using Biotype medium 1 as recommended by the scribed species, K. ascorbata and K. clyocrescens (2, 7, 17). manufacturer. The strips were incubated at 30°C for 4 days, and growth was Kluyvera strains have been isolated from both soil and clinical m TABLE 2. Biochemical reactions that differentiate DNA groups 1 through 11 - ~~ ~ ~ ~~ ~ ~ DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA group 1 group 2 group 3 group 4 group 5 group 6 group 7 group 8 group 9 group 10 group 11 (B. agrestis) (B.ferruptiae) (B. gaviniae) (B. hrennerae) (B. izardzi) (B.noackiae) (B. wunnnholdzue) (K. cochleue) (K.georgiaria) (K. ascorbatn) (K. ctyocrescetis)

v: .-2 z 2 .- C C c .- .3 =f g .- .- .- g g Test Method(s) used” w in 2 g 2 in w in in in LL L .-ca OhRr- ’c1 c ’* 0 II ’I O 11 O 11 0 11 $5 g.5 8.5 $5 Acetate Ut. (FGH) - 78 + 100 + 80 - 40 + 100 + 100 + 100 + 100 100 100 + 100 N-Acetyl-D-galactosamineh Ut. (FGH) + 94 + 100 + 70 + 100 + 100 + 93 + 100 - 0 60 67 + 100 N-Acetyl-L-glutamateh Ut. (FGH) + 94 + 100 + 90 + 100 + 100 + 100 + 100 - 0 0 0 - 0 N-Acetyl-L-glutamine Ut. (FGH) - 6- 0-20- 0-8+ 80- 0- 0 0 67 0 N-Acetylglycin e Ut. (FGH) - 0- 0- 0- 0- 0- 0 - 0- 0 20 67 0 Adonitol Acid. (SM), Ut.(FGH, IP) - 0 - 0 + 65 + 60 - 8 - 0 - 0- 0 0 0 - 0 D-Arabinosc’ Ut. (FGH) + 69 - 0 - 45 - 0 + 100 + 73 + 100 - 0 20 33 +- 67 D- Arabitol Acid. (SM), Ut. (FGH, IP) - 0 - 0 + 65 + 60 - 8 - 0 - 0- 0 0 0 0 L-Arginine dihydrolase” Alcal. (FGH, SM) - 0 - 0 + 100 + 70 - 15 + 100 + 100 - 0 20 0 - 0 L-Arginine Ut. (FGH) - 0- 0- 0- 0- 0- 20 - 0- 0 0 67 - 0 Citrate utilization (Simmons)” Ut. (FGH, SM, IP) + 50 - 0 - 36 + 60 + 100 + 100 - 0+100 80 96 - 80 rn-Coumarate(MI) Ut. (IP) - 0 - 0 - 0 - 0- 0ND”DND ND - 0 100 73 -+ 100 Dulcitol’ Aad. (FGH, SM), Ut. - 10 - 0 - 20 - 0 - 0 - 0 - 0+100 40 45 - 0 (FHG, IP) L-Fucose” Acid. (SM), Ut. (FGH, 1P) + 80 - 0 - 60 - 0 + 85 - 7 + 100 - 0 - 20 10 + 44 Gentisate (API)h Ut. (IP) - 0- 0- 1- 0- ONDNDND ND - 0 + 60 100 - 0 Glycerol” Acid. (SM) + 60 - 0- 0- 0- 0- 0 - o+100 25 40 - 5 3-Hydroxybenzoate“ Ut. (FGH, IP) - 0- 0- 0- 0- 0- 0 - 0- 0 + 100 80 - 0 3-Hydroxyphenylacetate” Ut. (FGH) - 0+ 75- 0- 0- 0- 0 - 0- 0 + 60 + 100 + 100 Indole production” Kovacs (SM) - 0- 0-30- 0- 0- 40 - 0- 0 + 100 + 92 + 90 nzyo-InositoP Acid. (FGH, SM) - 0- 0- 0- 0-0- 0+100- 0 0 - 0 - 0 KCN~ Growth (SM) + 90 + 100 + 82 + 100 + 100 + 100 - 0+ 100 + 100 + 92 + 86 5-Ketogluconateh Ut. (IP) - 10 - 0 + 97 + 100 - 50 + 93 - 0+100 + 100 + 100 + 100 Lactose” Acid. (SM) + 100 - 0 - 1 + 100 + 100 - 0 - 0+ 67 + 80 + 98 + 95 LactuIose” Acid. (FGH), Ut. (IP) - 10 - 0 - 0 - 0 - 0 - 0 - 0- 0 - 60 + 95 + 50 L-Lysinc decarboxylase” Alcal. (FGH, SM) - 0+100- 0- 0-0- 0- 0- 0 + 100 + 97 - 23 Malonate utilization” Ut. (FGH, SM) + 96 - 0 - 100 + 100 + 100 + 93 + 100 + 100 - 60 + 96 + 86 Maltitolh Acid. (FGH), Ut. (IP) + 75 + 100 + 88 + 100 + 30 + 87 - 0 + 67 + 100 + 100 + 100 Melibioseh Acid (SM) +55+ 75- I+90+50- 0- 0+ 100 + 100 + 99 + 100 u-Methyl-D-glucopyranoside” Acid. (SM), Ut. (IP) - 6- 25- 2- 40-0- 0- 0+100 + I00 + 100 + 100 3-Met hyl-~-glucopyranoside”Ut. (FGH, IP) + 95 + 75 + 95 - 40 + 85 + 87 - 0- 0 - 0 - 5 - 0 Methyl red Acid (SM) + 100 + 100 + 100 + 100 + 100 + 100 + 100 - 0 + 100 + 100 + 100 Mucateh Acid. (SM) + 71 - 25 - 55 + 100 - 30 + 100 - 0+100 + 100 + 90 + 81 L-Ornithine decarboxylasc” Alcal. (FGH, SM) + 100 + 100 - 0 - 30 + 100 - 7 - 0+100 + 100 + 100 + 100 Palatinose” Acid. (SM), Ut. (IP) + 98 + 100 + 100 + 100 - 40 + 100 - 0+100 + 100 + 100 + 100 Phenylacetateh Ut. (FGH, IP) - 60 + 50 - 20 - 0-70- 30 - 0+ 100 60 + 100 + 100 1.-Phenylalanine deaminase FeCI, test (SM) - 0- 0- 0- 0-0- 33 - 0- 0 0 - 0 - 0 3-Phenyl ropionatc” Ut. (FGH, IP) - 0- 0- 0- 0- 0- 0 - 0- 0 100 - 30 - 40 L-ProlineS1 Ut. (FGH, IP) + 80 - 75 + 98 + 100 + 100 + 80 + 100 - n 20 - 50 - 60 Raffinose” Acid. (SM), Ut. (IP) +28- 25- 2+90-0- 0- 0+ 100 100 + 98 + 100 D-Sorhitolh Acid. (SM), Ut. (1P) -60+100-25- 0-0- 0- o+67 0 + 40 - 45 Sucrose” Acid. (SM) - 2- 0- 14- 0-0- 0- 0- 33 100 t 98 + 81 u-Tagatose” Acid. (FGH, SM), Ut. - 10 - 0-20- 0- 0- 0 - 0+100 40 - 45 - 0 (FGH, IP) Tartrate (Jordan’s)” Acid (SM) + 60- 0 + 30 - 15-20+ 90 - 0 - 0 0 0 - 0 Vogcs-Proskaucr Acetoin (SM) - 0- 0- 0- 0- 0- 0 - 0+100 0 0 - 0 Ut., utilization test; Acid., acidification test; Acal., alcalization test.The information in parentheses indicates at which institutions the tests were performed, asfollows: FGH, Fachgebiet Hygiene, Technische Universitat, Berlin, Germany;SM, Staatliches Medizinaluntersuchungsamt,Braunschweig, Germany: IP, Institut Pasteur, Paris, France. ’ Test useful in differentiating DNA groups 1 through 11, which correspond to Buttiauxeffuand Kluyvera genomospecies. ND, test not done. 56 MULLER ET AL. INT.J. SYST.BACTERIOL.

TABLE 3. Logarithms of probability distances and relative binding ratios

Logarithm of probability distances or relative binding ratio at 60°C" DNA group DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA group 1 group 2 group 3 group 4 group 5 group 6 group 7 group 8 group 9 group 10 1 50.60 55.64 53.33 62.67 58.00 56.00 2 23.92 50.2 49.83 48.33 55.00 49.00 3 22.28 34.50 60.16 52.40 70.50 48.71 4 23.42 24.65 23.05 52.56 55.00 49.25 5 15.46 26.94 19.44 16.21 50.29 48.00 6 24.77 31.60 16.77 24.31 16.93 47.92 7 35.99 33.59 30.89 33.61 26.60 27.86 8 26.65 33.47 40.15 31.43 36.52 43.76 50.90 9 30.57 35.89 39.04 35.43 36.22 40.66 50.19 28.28 10 28.82 36.41 39.83 41.42 39.95 40.01 57.77 29.73 10.79 11 23.16 26.39 35.26 30.47 28.97 34.86 43.35 26.32 13.77 14.88

'' The values on the lower left are logarithms of phenotypic probability distances; these values were calculated by using all of the data in Table 2 except the m- coumarate and gentisate data. The values on the upper right are relative binding ratios at 60°C (expressed as percentages); these values were obtained from Table 6.

sources, although soil, water, and humans do not seem to be group 6, represented by the unnamed species enteric group 59, the normal habitats of Kluyvera species (14, 15). contained 15 snail and slug strains. Our experience indicates that strains of both genera occur None of the Kluyvera-like strains hybridized at the species frequently and abundantly in the intestines of snails, slugs, and level to members of hybridization group 10 (K. ascorbata), other mollusks. The apparent diversity of the different Butti- hybridization group 11 (IC clyocrescens),or hybridization group 9 auxella and Kluyvera species isolated from mollusks is an ad- (unnamed Kluyvera species 3 [ll, 141). Three Khyvera-like ditional reason to consider these animals to be the ecologic strains isolated from snails and slugs belonged to hybridization niche and natural source of these bacteria. group 8. Essentially without exception, the levels of relatedness DNA relatedness. The results of our DNA-DNA relatedness within each hybridization group were more than 70% at both experiments revealed that there were 11 DNA hybridization the optimal temperature (60°C) and the stringent temperature groups or genomospecies (Table 6). Eighteen snail and slug (75°C) used for DNA reassociation (Table 6). Similarly, essen- isolates and the type strain of B. agrestis were in hybridization tially without exception, the levels of divergence within related group 1. None of the mollusk isolates were in hybridization sequences were 5% or less. These levels of relatedness corre- group 2, which corresponds to the unnamed Buttiauxella spe- spond to the levels recommended for strains belonging to a cies called enteric group 63; this was due to the screening tests single species (19). used during isolation. The strains isolated were distinguished The levels of relatedness obtained for members of hybrid- on the basis of a negative lysine decarboxylase test. Since ization groups 1 through 7 were generally between 45 and 65%, enteric group 63 strains are positive for this reaction, if they a good indication that all of these groups belong to a single were present, they would have been eliminated by the screen- genus, the genus Buttiauxella. The hybridization groups that ing procedure used. Hybridization group 3, represented by exhibited the highest levels of relatedness were hybridization enteric group 64 containing one previously described strain, groups 1 and 5 and hybridization groups 3 and 6. The members contained 35 snail and slug strains. Hybridization groups 4, 5, of hybridization group 8 were 44 to 48% related to the type and 7 each represented previously unrecognized species con- strains of the two previously described and one previously taining seven, nine, and five strains, respectively. Hybridization unnamed Kluyvera species, but only 13 to 25% related to the

TABLE 4. Results obtained when DNA groups were compared

Comparison No. of Phenotypic probability distance DNA-DNA relatedness value tests (mean ? SD) (mean ? SD)

Within the genus Buttiauxella 21 25.37 t 6.31 53.49 t 5.69 DNA group 1 vs DNA groups 2 through 7 6 24.31 t 6.63 56.04 t 4.12 DNA group 2 vs DNA groups 1 and 3 through 7 6 29.20 t 4.62 50.49 ? 2.36 DNA group 3 vs DNA groups 1, 2, and 4 through 7 6 24.49 ? 6.83 56.27 2 8.09 DNA group 4 vs DNA groups 1 through 3 and 5 through 7 6 24.21 t 5.56 53.36 t 3.97 DNA group 5 vs DNA groups 1 through 4, and 7 6 20.26 2 5.22 52.38 % 5.40 DNA group 6 vs DNA groups 1 through 5 and 7 6 23.71 2 5.92 56.12 2 7.93 DNA group 7 vs DNA groups 1 through 6 6 31.42 t 3.65 49.81 ? 3.08

Buttiauxella DNA groups vs Kluyvera DNA groups 28

Within the genus Kluyvera 20.63 t 8.37 DNA group 8 vs DNA groups 9 through 11 26.55 2 2.55 DNA group 9 vs DNA groups 8, 10, and 11 17.61 t 9.36 DNA group 10 vs DNA groups 8, 9, and 11 18.47 2 9.97 DNA group 11 vs DNA groups 8 through 10 18.32 ? 6.95 VOL.46, 1996 NEW BUTTIAUXELLA AND KLUYVER.4 SPECIES 57

members of the seven Buttiamella hybridization groups, which $ is consistent with placement of hybridization group 8 in the genus Kluyvera. The levels of relatedness between members of 6 the Buttiamella and Kluyvera hybridization groups and more than 50 other species belonging to the Enterobacteriaceae were Buttiauxelfa generally between 15 and 30% (data not shown). Certain strains included in the hybridization groups exhib- Kluyvera ited higher levels of divergence (more than 5%) or lower levels of relatedness (less than 70% in 60°C reactions or less than Budvicia 60% in 75°C reactions) than the levels used in the definition of a genetic species (19). In almost all cases, these borderline strains deviated in only one of the three parameters used to Cedecea determine species level relatedness, and their levels of relatedness with a second reference strain were fully within the Citrobacter species definition. For example, EG59-like strain NSW 155 in hybridization group 4 was 66% related to the reference strain Edwardsiella at 60°C but exhibited only 0.5% divergence and the level of relatedness at 75°C was 66%. Similarly, strain S1/3-1052 in Enterobacter hybridization group 3 exhibited 5.5% divergence with one reference strain, but 5.0% divergence with the second reference Escherichia and Shigella strain, and, in both cases the levels of relatedness at both temperatures were well within the limits used to define species. In every case, all of the strains belonging to a relatedness group Ewingella exhibited similar biochemical reactions. Therefore, we decided that they should be included in the relatedness group, at least Hafnia for the present. Phenotypic and chemotaxonomic characterization. All of the Klebsiella strains studied were gram-negative, oxidase-negative, catalase- positive, D-glucose-fermenting, nitrate-reducing, rod-shaped 1,eclercia organisms belonging to the family Enterubacten’aceae. They grew on MacConkey agar and on Endo agar at 30°C. With few L,erninorella exceptions, all of the strains were positive in standard tests for motility, acid and gas production from D-glucose, fermentation of D-mannitol, salicin, L-arabinose, L-rhamnose, maltose, D- Moellerella xylose, trehalose, cellobiose, D-mannose, galactose, gentiobiose, D-ribose, and arbutin, esculin hydrolysis, and o-nitrophenyl-P- Morganella D-galactopyranoside (P-galactosidase) test. They were negative for production of hydrogen sulfide on triple sugar iron, Chris- Obesumbacterium tensen urease activity, gelatin liquefaction, lipase activity (Tween 80 and corn oil), DNase activity, and production of a Pantoea yellow pigment or some other pigment and did not ferment 2-deoxy-~-glucose,2-deoxy-~-ribose, erythritol, a-D-fucose, D- Pragca lyxose, and xylitol. Almost all of the strains utilized the following compounds as D- I’roteus sole carbon and energy sources: N-acetyl-D-glucosamine, alanine, L-alanine, L-arabinose, arbutin, DL-asparagine, D-cellobiose, D-fructose, D-galactose, D-galacturonate, gentiobiose, Provideticia D-gluconate, D-glucosamine, D-glucuronate, L-glutamine, L-glutamate, DL-glycerate, glycerol, 4-hydroxybenzoate, 2-ketoglu- R~hnella conate, D-lactate, DL-lactate, maltose, maltotriose, mannitol, D-mannose, methyl-a-galactoside, methyl-P-galactoside, meth- Salmonella yl-P-D-glucoside, mucate, oxaloacetate, palatinose, protocatechuate, pyruvate, quinate, L-rhamnose, D-ribose, D-saccha- Serratia rate, salicin, L-serine, starch, D-trehalose, and D-xylose. Almost all of the strains were not able to utilize the following Tatumella compounds as sole carbon and energy sources: acetamide, acetamidocaprate, N-acetyl-DL-methionine, N-acetyl-L-proline, cis-aconitate, trans-aconitate, adipate, p-alanine, allantoin, al- Trabulsiella trose, ~~-2-aminoadipate,2-aminobenzoate, 3-aminobenzoate, 4-aminobenzoate, ~~-2-aminobutyrate,~~-3-aminobutyrate, 4- Xenorhabdus aminobutyrate, ~~-2-amino-isobutyrate,~~-3-amino-isobutyrate, 5-aminovalerate, aminoxyacetate, amygdalin, anthranilate, Yersinia 2-hydroxy-phenylacetate,L-arabitol, arabonate, D-arginine, D- asparagine, azelate, benzoate, betaine, 1-butanol, 2-butanol, Yokenella n-butyrate, isobutyrate, cadaverine, caprate, caprylate, carni- tine, carnosine, L-citrulline, citraconate, L-cysteinate, dextran, TABLE 6. Identification of Buttiamella and Kluyvera strains by DNA-DNA hybridization

Source of labeled DNA

Source of DNA group 1 strains DNA group 2 DNA group 3 strains DNA group 4 strain? DNA group 5 strains DNA group 6 ctrains DNA group DNA group unlabeled strain CDC strain strain DNA 1180-81T NSW 3XT S3i1-49T CDC 1176-8IT S313-162 (serial CDC 1175-81 SllI-984T 2 2132 S1l6-57lT S3i8-879 S312-l6lT NSW 155 NSW 1IT CDC 1650-80 (serial (serial (serial no. 001) (serial no. 012) no. 054) (serial no. 058) (serial no. 062) (serial no. 143) (serial no. 145) (scrial no. 149) (serial no. 151) (scrial no. 161) (serial no. 170) (serial no. 179) no, 182) no. 185)

RBR RBR RBR RBR RBR RBR RBR RBR RBR RBR RBR RBR RBR RBU RBU RBR RBR DNA Serial RBR RBR RBR RBR RBR RER UBR RBR RBR RBR Strain at ~h at at at at at at at at at at at at at at at at at at at at at at at at at at group no. D D D D D D D D D D D D D 60°C 75°C 60°C 75°C 60°C 60°C 75°C 60°C 75°C 60°C 75°C 60°C 75°C 60°C 75°C 60°C 75°C 60°C 75°C 60°C 75°C 60°C 75°C 60°C 75°C 60°C 75°C

I 001 CDCl 176-8IT 100 0 100 78 3.5 68 52 i2.n 54 10.5 57 11.0 36 64 1n.u 28 56 90 32 56 95 31 59 6.5 36 66 65 48 52 64 11.5 27 56 24 16.0 012 S313- I62 75 5.0 62 100 0 100 53 11.0 ND' ND ND ND ND 62 7.0 43 ND ND ND ND 18 17.0 013 S313-203 74 7.0 60 85 2.5 84 ND ND ND ND ND ND 61 6.5 ND ND ND ND 21 16.5 017 S3i4-776 78 5.5 67 93 1.5 92 ND ND ND ND ND ND 61 5.0 ND ND ND ND 21 15.5 018 s3i4-779 78 5.0 69 91 0.5 92 ND ND ND ND ND ND 65 5.1 ND ND ND ND 23 16.0 021 s315-201 71 5.0 63 90 1.0 88 ND ND ND ND ND ND 62 5.0 ND ND ND ND 19 15.5 022 s315-757 75 5.0 63 75 4.9 69 ND ND ND ND ND ND 60 7.0 ND ND ND ND 25 18.5 023 S316-333 84 3.0 78 74 5.5 68 ND ND ND ND ND ND 6n 7.0 ND ND ND ND 024 S317-896 78 5.0 63 89 2.0 85 ND ND ND ND hD ND 67 5.5 40 ND ND ND ND 1918 18.017.5 025 s317-897 77 4.5 66 90 2.0 88 ND ND ND ND ND ND 68 5.5 ND ND ND ND 16 17.5 026 S3i8-910 73 5.0 62 85 2.0 84 ND ND ND ND ND ND 60 5.5 42 ND ND ND ND 18 17.0 029 S3i9-878 83 4.0 76 78 4.0 70 ND ND ND ND ND ND 61 6.5 ND ND ND ND 19 17.5 030 s3i9-887 83 4.5 74 77 4.0 67 ND ND ND ND ND ND 60 8.2 ND ND ND ND 16 18.0 n3 I S3110-769 75 7.5 58 83 3.0 79 ND ND ND ND ND ND 60 7.5 ND ND ND ND 13 18.0 032 S3111-533 87 4.0 81 76 5.0 68 NI) ND ND ND ND ND 6n 75 m ND ND ND 20 17.0 033 S3i11-794 75 4.5 67 73 5.5 65 ND ND ND ND ND ND 57 7.0 ND ND ND ND 23 16.5 037 s3114-1071 79 4.5 66 78 5.0 66 ND ND ND ND ND ND 62 6.5 ND ND ND ND 24 16.0 042 s3i14-1085 75 5.0 66 75 5.5 64 ND ND ND ND ND ND 62 6.5 ND ND ND ND 23 15.0 053 Slil3-692 73 5.5 66 ND 54 11.5 52 11.0 51 12.0 28 ND 55 80 53 8.5 30 ND ND ND ND ND ND

2 054 CDC 1180-8IT 46 11.0 25 48 9.0 inn o 44 9.5 47 11.5 26 54 9.5 24 52 90 13 48 100 25 43 7.5 46 90 20 52 58 9.0 23 49 19 15.0 m 3 058 CDC 1175-81 57 12.0 32 58 10.0 56 10.0 100 0 100 88 0 89 85 3.5 74 63 65 14 64 7.0 4s 49 8.0 55 95 22 71 6.5 54 77 6.5 60 54 24 16.0 062 Slll-984T 53 11.5 29 55 9.5 37 50 12.5 87 2.5 86 100 n ion ND 61 70 12 ND 50 85 ND ND ND ND 20 15.5 nm Slil-986 ND ND ND 91 2.0 89 100 1.0 ion ND ND ND ND ND ND ND ND ND 085 S112-994 ND ND ND 90 3.5 82 100 40 ion ND ND ND ND ND ND ND ND ND 087 SIi2-1111 ND ND ND 89 2.0 86 86 2.0 87 ND ND ND ND ND ND ND ND ND -- 088 SI 13-1052 ND ND ND 80 3.3 67 77 5.0 69 ND ND ND ND ND ND ND ND ND 089 SI13-1200 ND ND ND 76 3.5 66 83 5.0 73 ND ND ND ND ND ND ND ND ND 090 Sli4-695 55 ins 36 56 100 ND 89 0.5 88 86 2.0 88 ND ND ND ND ND ND ND ND ND 093 s1i5-857 ND ND ND 78 4.0 67 75 53 70 ND ND ND ND ND ND ND ND ND 094 SIi5-860 ND ND ND 75 5.0 64 76 5.0 70 ND ND ND ND ND ND ND ND ND 095 S117-674 ND ND ND 77 5.5 73 74 6.5 65 ND 44 I00 ND ND ND ND ND ND ND 09 6 SU7-I 143 ND ND ND 80 7.0 74 84 3.5 79 ND ND ND ND ND ND ND ND ND 099 s1i9-514 ND ND ND 76 7.5 76 77 2.5 75 ND ND ND ND ND ND ND ND ND I00 SliY-518 NI) ND ND 80 2.5 78 80 3.0 78 ND N 1) ND ND ND ND ND ND ND 103 s1110-801 ND ND ND 80 1.0 78 SO -3.0 76 ND ND ND ND ND ND ND ND ND 104 s1110-853 ND ND N 1) 82 2.5 17 79 5.0 72 ND ND ND ND ND ND ND ND N 1) 105 S 1110-863 ND ND ND 83 2.0 18 78 2.0 78 ND ND ND ND ND ND ND ND ND 108 Slill-577 ND ND ND 85 1.5 77 83 15 89 ND NV ND ND ND ND ND ND ND 110 Slill-707 ND ND ND 81 2.0 71 86 2.5 75 ND ND ND ND ND ND ND ND ND 113 Slil2-lI80 ND ND ND 87 7.0 74 79 3.0 76 ND ND NV ND ND ND ND ND ND 114 Sli14-564 ND ND ND 84 2.0 17 80 2.0 8 ND ND ND ND ND ND ND ND ND I15 S1114-669 ND ND ND 88 2.0 79 81 3.5 82 ND ND ND ND ND ND ND ND ND I I9 Slil5-774 ND ND ND 87 2.5 75 84 4.0 78 ND ND ND ND ND ND ND ND ND 121 Sli15-828 ND ND ND 84 1.0 82 87 2.0 83 ND ND ND ND ND ND ND ND ND 123 S 1116- 12 ND ND ND 72 4.5 52 77 7.5 63 ND 55 90 ND ND ND ND ND ND ND 124 Slil7-7 ND ND ND 86 1.0 80 1.0 83 ND ND ND ND ND ND ND ND ND 129 S1117-911 ND ND ND 89 I .o 84 87 1.0 86 ND ND ND ND ND ND ND ND ND I30 S1118-662 ND N I> ND Xh 2.0 $2 85 1.5 83 ND NV ND ND ND ND ND ND ND 132 Sli18-784 ND ND ND 87 2.0 78 87 I 5 8.5 ND ND ND ND ND ND ND ND ND 134 S li 19-66] ND ND ND 86 2.0 77 86 :.(I 82 ND ND ND ND ND ND ND ND ND 116 sui 9-76n ND ND ND s5 1.5 79 86 1.5 83 ND ND ND ND ND ND ND ND ND 139 E(i59-like strain K 2258 ND ND ND ND ND 81 3.0 70 ND ND ND ND 74 5.5 56 70 6.5 50 ND ND 140 EG59-like strain K 2286 ND ND NI> ND ND 74 30 67 ND ND ND ND 67 5.0 51 69 6.0 47 ND ND I41 EGWlike stiaiii K 2340 ND ND ND ND ND 73 3.0 06 ND ND ND ND 71 4.5 56 69 5.5 52 ND ND 132 EG59-liks strilin SA 1191 ND ND ND ND ND 79 3.0 70 ND ND ND ND 69 5.0 53 61 7.5 39 ND ND 143 EGS9-like strain Z 7132 ND ND ND ND ND 100 100 60 59 5.5 35 52 51 70 5.5 51 70 7.0 50 50 ND 4 144 S116-360 ND ND ND 61 1.5 65 9.0 48 ND 89 15 86 ND ND ND ND ND ND ND 145 51/6-571T 57 11.5 32 58 9.5 37 57 11.5 64 7.5 65 8.5 4Y ND I on I) 100 84 1 5 81 5.3 80 57 61 ND 55 24 16.0 146 S1.6-706 ND ND ND 60 8.0 57 12.5 45 NU 88 25 78 ND ND ND ND ND ND ND 147 S118-1022 ND ND ND 64 7.0 63 10.0 46 ND 89 3.5 77 ND ND ND ND ND ND ND 148 5118-783 34 13.0 ND 34 13.5 43 9.0 39 17.0 26 ND 68 80 34 71 50 64 ND ND ND ND ND ND 149 S3/8-879 54 9.5 35 57 110 36 57 120 60 65 ND 68 8.0 82 I 5 84 I00 0 100 49 75 60 61 66 12.5 54 22 16.5 I50 S3112-695 ND ND 56 12 (1 63 6.5 ND ND ND 84 15 79 50 8.0 ND ND ND ND 22 16.0

5 151 S312-161T 66 I10 40 71 6.5 57 56 12.5 ND ND 50 11.5 60 S2 I00 0 lU0 92 0.5 78 60 b2 14.0 58 20 17.0 152 s3/2-199 59 105 42 ND ND ND ND ND ND ND 97 0.5 Yh ND ND ND ND 21 16.5 158 S3110-487 67 95 46 ND ND ND ND ND ND ND 88 1.0 89 ND ND ND ND 24 165 15Y 53113-626 68 Y.U 47 ND ND ND ND ND ND ND 87 0.5 S5 ND ND ND ND 22 16.0 I60 EGS9-tikc wain NSW ND ND ND ND ND 55 10.0 29 ND ND ND IOU 0.5 ion 54 51 ND ND 150 161 EG59-like strain NSW ND ND ND ND ND 50 IOU 25 48 44 66 0.5 66 100 0 IOU 45 50 46 ND IS5 162 EGSY-Iikc strain RA ND ND ND NU ND 49 ND ND ND 74 2.0 70 46 49 ND ND 144 163 EG59-like strain RA ND ND ND ND ND 54 ND ND ND 79 1.5 74 51 53 ND ND 151

ND ND ND 6 164 EGS9-like strain BR ND ND ND ND ND ND 85 15 XU 85 1.0 80 ND ND 489 ND ND ND 165 EGSY-like strain BR ND ND ND ND ND ND ND 86 15 81 ti5 30 84 ND ND 53Y ND ND ND ND ND 166 EG59-like wainBr 588 ND ND ND ND ND ND 8.5 1.5 80 82 3 0 76 ND ND ND ND ND ND 167 EG5Y-like strain BR ND ND ND ND ND 82 1.5 78 85 2.5 82 ND ND 607 ND ND ND ND ND 168 EG59-like wain BR ND ND ND ND ND 100 0.5 100 86 2.0 83 ND ND 626 ND ND ND ND ND 16Y EG59-like wainBR ND ND ND ND ND 88 1.5 82 82 1.5 84 ND ND 637 ND ND ND ND 170 EG59-like wain NSW ND 70 70 57 54 50 45 4b 10.5 100 0 100 84 2.5 88 50 115 ND 1 iT ND ND ND 171 EG5Y-like main NSW ND ND ND ND ND ND ND 100 I0 90 83 2.0 78 ND ND 87 ND ND ND ND ND 172 EG59-like strain NSW ND ND ND ND ND 100 I u 100 8s 20 84 ND ND 120 ND ND ND I73 EG59-like strain NSW ND ND ND ND ND ND ND lo0 05 IIH) 85 2.0 82 ND ND 245 ND ND ND ND ND 174 EG59-likc wain NSW ND ND ND NU ND 87 1 5 84 86 1.S 83 ND ND ND 197 ND ND ND EG59-like strain NSW ND ND ND ND ND ND X3 I5 85 84 ND ND 175 ND 1.0 82 336 ND ND ND EG59-like wain NSW ND ND ND ND ND ND 9(1 1.0 87 87 1.5 ND 176 ND 85 ND ND 402 ND ND ND ND I77 EG59-like strainRA ND ND ND ND ND 83 1.5 82 81 1.5 80 ND ND 116 ND ND ND ND 178 EG59-like strain SA ND ND ND ND ND 88 IS 83 89 0.5 87 ND ND ND 1313 ND ND CDC lh50-80 (= EGSY) ND 73 60 57 55 38 47 4') 85 I5 86 I00 0 IN) 51 ND 179 ND ND ND 7 180 EG59-like strain NSW ND ND 42 ND ND ND 46 47 98 0.5 98 ND ND I84 ND ND 181 EG59-like strain NSW ND 47 ND ND ND 47 49 49 loo 0.5 99 ND ND 253 ND ND 1x2 EGS9-likc strain NSW ND ND ND 50 44 44 Ib 45 48 47 loo 0 100 ND 326' ND ND ND 183 EG59-like strain NSW ND ND 52 ND ND ND 50 46 49 I(K) 0.5 LOO ND 393 ND ND 184 EGSY-Iikc strain SA 470 ND ND 46 ND ND ND 46 46 47 98 0 94 ND ND ND 8 185 S311-49' 32 I73 23 15.5 19 160 ND ND ND ND _-77 12.5 ND ND ND ND 100 0 100 186 S311-913 24 17.0 25 1.5.0 ND ND ND ND ND ND 33 135 ND ND ND ND 92 1.0 90 187 5314-835 7-3 130 13 ND ND ND ND ND ND 14 13.5 ND ND ND ND 86 0.5 89 RBR, relative binding ratio, expressed as a percentage. D. level of divergence, expressed as a percentage. ' ND, test not done. 60 MULLER ET AL. INT. J. SYST.BACTERIOL.

~~-2,4-diaminobutyrate,diaminopimelate, 2,3-diaminopropio- mean values. The most typical DNA group could not be de- nate, dimethylglycine, m-erythritol, ethanol, ethanolamine, termined with certainty. Buttiauxella DNA group 5 exhibited ethylamine, D-fucose, D-glutamate, glutarate, glycinamide, gly- the closest phenotypic probabilistic distance with other Butti- cogen, glycolate, glycyrrhizinate, 1-hexanol, 1,6-hexandiol, auxella DNA groups; however, DNA groups 3, 6, and 1 were hexylamine, hippurate, histamine, D-histidine, L-histidine, L- the closest groups on the basis of DNA relatedness data. homoserine, ~~-3-hydroxybutyrate,4-hydroxybutyrate, ~~-2-hy- The mean value for six total phenotypic probability distances droxycaprate, ~~-2-hydroxy-isobutyrate,~-2-hydroxyisocaprate, in the genus Klcyvera was log 20.63, and the standard de- 2-hydroxy-isovalerate, DL-y-hydroxylysine,4-hydroxyphenylgly- viation was log 8.37. However, the mean value for 28 total cine, DL-hydroqpolhe, 2-hydroxyvalerate, HQ-P-glucuronide, phenotypic probability distances between the genera Buttiaux- heptanoate, indole-3-acetate, isophthalate, itaconate, 2-keto- ella and Kluyvera was log 37.06 (standard deviation, log 7.79). glutarate, 2-ketoisocaprate, D-leucine, L-leucine, levulinate, These high phenotypic probability distances indicate that there D-lysine, D-malate, maleate, D-mandelate, L-mandelate, are significant differences between the genus Bicttiauxella and D-mannoheptulose, D-melezitose, mesaconate, mesoxalate, the genus Kluyvera. Although these two genera have many D-me t hionine, L-me t hionine, DL-met hioninesulfone, methyl-a- similar phenotypic characteristics, the phenotypic data correlate D-glucoside, a-methyl-D-mannoside, g-methyl-D-xyloside, L- with the DNA hybridization data and indicate that they are sep- norleucine, D-norvaline, L-norvaline, 1,8-octandiol, D-orni- arate genera. thine, phenoxyacetate, D-phenylalanine, phenylglycine, phenyl- Previously reported conventional test results for Buttiamella lactate, phosphoenolpyruvate, phthalate, pimelate, poly-D- and Kluyvera strains are compared with the results obtained for galactomannan, polygalacturonate, D-proline, propionate, other genera belonging to Enterobacteriaceae in Table 7 (1 1, protocatechuate, putrescine, salicylamide, salicylate, sarcosine, 12, 13). Although some biochemical characteristics can be used sorbate, L-sorbose, spermine, suberate, D-tartrate, meso-tar- to differentiate various Buttiauxella and Kluyvera species (N- trate, tartronate, taurine, thiamine, tricarballylate, trigonelline, acetyl-L-glutamate, rn-coumarate, glycerol, 3-hydroxyphenyl- tropate, tryptamine, D-tryptophan, L-tryptophan, tyramine, L- acetate, indole production, lactulose, lysine decarboxylase tyrosine, ureidosuccinate, n-valerate, isovalerate, L-valine, xy- activity, melibiose, a-methyl-D-glucopyranoside, raffinose, su- litol, and L-xylose. crose) (Table 2), no single biochemical test result differentiates The strains varied in their ability to utilize the following these genera with 90 to 100% certainty. Nine test results (in- compounds as sole sources of carbon and energy, and these dole production, citrate utilization, L-arginine dihydrolase ac- characteristics could not be used to differentiate hybridization tivity, ornithine decarboxylase activity, and fermentation of groups: D-alanine, L-asparagine, L-aspartate, benzoate, fumar- lactose, sucrose, raffinose, or-methybglucopyranosidase, and ate, D-glucarate, D-glucosaminate, glycerate, glycerophosphate, melibiose) do give differences of 40 to 50% between Buttiailx- glycine, glyoxylate, 4- hydroxybenzoat e, 4-hydroxyphenylace- ella and Kluyvera species. The phenotypic probability distances tate, inulin, DL-isocitrate, DL-isoleucine, 2-ketoglutarate, L-lac- between Buttiauxella and Kluyvera species and other genera tate, D-lyxose, L-lyxose, L-malate, L-mannose, phenylpyruvate, that belong to the Enterobacteriaceae are shown in Table 5. D-serine, spermidine, succinate, L-tartrate, L-threonine, and D- These data are not identical to the data in Table 3 because they turanose. were calculated by using 47 conventional tests (12). The lowest The results of 44 biochemical tests that were used to differ- probability distance (log 12.46) was the distance between the entiate the 11 DNA hybridization groups belonging to the genus Buttiauxella and the genus Enterobacter. The probability genera Buttiauxella and Kluyvera in our numerical taxonomic distance between the genus Kluyvera and the genus Enter- study are shown in Table 2. The most useful tests for differ- obacter was log 15.33. It will be interesting to determine entiating the 11 genomospecies with binary phenetic data are whether the probability distances correlate with relatedness indicated. Hybridization groups 1 and 5 could not be distin- trees generated from rRNA sequence similarity data. guished on the basis of the results of any single test. These On the basis of the biochemical and DNA hybridization groups had to be distinguished on the basis of their overall data, we concluded that the genus Buttiamella contains the biochemical profiles. Hybridization groups 3 and 6 could not following seven species: B. agrestis, species corresponding to be separated on the basis of the results of a single test, but previously described enteric groups 59, 63, and 64, and three could be differentiated on the basis of their N-acetyl-L-glu- previously undescribed species. The genus Kluyvera contains tamine, adonitol, D-arabitol, L-fucose, mucate, and tartrate re- four species, K. ascorbata, K. cryocrescens, a species corre- actions. As stated above, these two pairs of hybridization sponding to Kluyvera unnamed species 3 (ll),and a previously groups exhibited the highest levels of interspecies DNA relat- undescribed species. Taxonomic proposals to name and define edness. the new species and to emend the description of B. agrestis are The probability distances that separated pairs of hybridiza- given below. tion groups when the biochemical reaction results shown in Emended description of Buttiauxella agrestis Ferragut, Izard, Table 2 were used are shown in Table 3. The probability Gavini, Lefebvre, and Leclerc 1981. B. agrestis was called DNA distances for the 11 DNA hybridization groups varied from log hybridization group 1 in this study. The original description 10.79 to log 57.77. The lowest probability distances between (13), which was based on the 17 strains originally described by hybridization groups in the same genus were log 15.46 for Gavini et al. (15), stated that 60% of B. agrestis strains were Buttiauxella hybridization groups 1 and 5 and log 10.79 for malonate positive; we found (Table 2) that 96% of the strains Kluyvera hybridization groups 9 and 10. The intergeneric prob- were malonate positive. The biochemical tests that are useful ability distances ranged from log 23.16 to log 57.77. Table 4 for differentiating B. agrestis from other Buttiamella species are shows that the mean value for 21 phenotypic probability dis- the arginine dihydrolase, fucose, glycerol, lactose, melibiose, tances in the genus Buttiauxella was log 25.37 (standard devi- ornithine decarboxylase, palatinose, and D-sorbitol tests. This ation, log 6.31). The corresponding value for 21 DNA-DNA species has been isolated from mollusks, as well as water, soil, relatedness pairs (based on the relative binding ratios at 60°C and humans. shown in Table 6) was 53.49% (standard deviation, 5.69%). Description of Buttiauxella ferragutiae sp. nov. Buttiauxella The phenotypic and genomotypic values for DNA groups 7 ferragutiae (fer'ra.gut.i.ae. M. L. fem. gen. n.ferragutiae, of Fer- and 2 correlate well; these values are the farthest from the ragut, in honor of Carmen Ferragut, a French microbiologist, VOL. 46, 1996 NEW BUTTUUXELLA AND KLUY‘VERA SPECIES 61

Buttiauella

Kluyvera

Budvicia

Cedecea 2 w Citrobacter 0;: 0 Edwurdsiella

Enterobacter

Escherichiu and Shigellu

Ewingella

Hafnia I 5‘

Klebsiellu

U Leclercia E. s. L c3 i e i CD 000000C001A130000000~003800000&000~8000000000000000 04 owl wl Liminorella z E. c CL c-’ i i- c i 80~01300s%0~28000000~08000800ooo0000000020 Moellerella a

Motganella

L r c- 00wl0008000~00000000~~~~0000000C000000000000C0CCwl0 0 00 Y Obesumbacterium

Pantoea

Prugia

Proteus

Providencia

Rahnella

Salmonella

Serratia

e c c a Tatuniella 8000~0000040~0000~~00~0000%00~0C80000000000Nwl00

ice c +i+cc +- i -i--ci L Y 8080800~s8000~008888808800~0800~808088s8008~08~Trabulsiella

i rd N w Xenorrha bdus s~004404~03o0c00000030000000000c0f204a80000co0w000

Yersiniu

Yokenella 62 MULLER ET AL. INT.J. SYST.BACTERIOL. for her contribution to the study of the genus Buttiuuxella [13]) tion of several characteristics differentiates these taxa. Useful was previously called enteric group 63 (10,12) and in this study differential biochemical tests are the arabinose, citrate, fucose, was referred to as DNA hybridization group 2. Biochemical myo-inositol, ornithine decarboxylase, and raffinose tests. Iso- characteristics are shown in Table 2. Positive lysine decarbox- lated from mollusks. ylase and D-sorbitol tests and negative ketogluconate and ma- Description of the type strain. B. izurdii type strain S3/2-161 lonate tests clearly differentiate this species from all other (= ATCC 51606 = DSM 9397 = serial no. 151) was isolated Buttiuuxellu species, Isolated from water and soil. Because of from a snail sampled in Braunschweig, Germany. Its colonies the screening procedure, any strains present in mollusks would are circular, convex, greyish, and smooth on nutrient agar and have been discarded; therefore, it is not known whether this blood agar. They are 1 to 2 mm in diameter after 1 day and 2 species occurs in mollusks. to 3 mm in diameter after 2 days at 30 to 36°C. Weak, slow Description of the type strain. The type strain of B. ferrugu- growth occurs at 42°C. The type strain has all of the biochem- tiue is strain CDC 1180-81 (= CUETM 78-31 = ATCC 51602 ical characteristics given for the genus in Table 7 and for the = DSM 9390 = serial no. 054). This strain was isolated from species in Table 2. unpolluted soil (13). Its colonies are circular, convex, greyish, Description of Buttiauxella noackiae sp. nov. Buttiuuxellu no- and smooth on nutrient agar and blood agar. They are 1 to 2 uckiue (no.ack’i.ae. M. L. fem. gen. n. noackiae, of Noack, in mm in diameter after 1 day and 2 to 3 mm in diameter after 2 honor of Katrin Noack, who did most of the physiological tests days. Good growth occurs at 30 to 36°C. Weak, slow growth oc- at the Technische Universitat Berlin and spent many years curs at 42°C. Biochemical characteristics are shown in Table 2. studying the usefulness of miniaturized automated tests for Description of Buttiauxella gaviniae sp. nov. Buttiuuxellu differentiation of various bacterial groups) was previously guviniae (ga.vin’i.ae. M. L. fem. gen. n. guviniue, of Gavini, in called enteric group 59 (10, 12) and in this study was referred honor of FranCoise Gavini, a French microbiologist, for her to as DNA hybridization group 6. Biochemical characteristics contributions to the study of the genus Buttiuuxellu [14, 151) was previously called enteric group 64 (10, 12) and in this study of 15 strains isolated from snails are shown in Table 2. These was referred to as DNA hybridization group 3. Biochemical characteristics are almost identical to those described for en- characteristics are shown in Table 2. Positive arginine dihydro- teric group 59; the only exception is the result of the lactose lase, 5-ketogluconate, and palatinose reactions and negative test. All snail isolates lack a yellow pigment and are lactose ornithine decarboxylase and raffinose reactions are useful in negative, but seven of the eight enteric group 59 strains iso- differentiating B. guviniae from other Buttiuuxellu species. A lated from humans and food were lactose positive (10, 12). It combination of reaction results is necessary to differentiate this is possible that there is a correlation between the ability to split species from Buttiuuxellu nouckiue. All but one strain were and metabolize lactose and the ability to survive in humans. isolated from mollusks. Tests that are useful in differentiating B. noackiue from other Description of the type strain. B. guviniue type strain S1/1- Buttiuuxellu species are the N-acetyl-L-glutamine, L-arginine 984 (= ATCC 51604 = DSM 9393 = serial no. 062) was iso- dihydrolase, melibiose, and L-ornithine decarboxylase tests. In lated from a snail sampled in Braunschweig, Germany. Its addition, 33% of the strains are phenylalanine deaminase pos- colonies are circular, convex, greyish, and smooth on nutrient itive and 40% produce indole. A combination of several char- agar and blood agar. They are 1 to 2 mm in diameter after 1 day acteristics is necessary to distinguish B. nouckiue from other and 2 to 3 mm in diameter after 2 days at 30 to 36°C. Grows Buttiuuxellu species. Isolated from mollusks, human sputum, poorly at 42°C. The type strain has all of the characteristics human wounds, and food. It is not known whether this species given for the genus in Table 7 and for the species in Table 2. is pathogenic. Description of Buttiauxella brennerae sp. nov. Buttiuuxellu Description of the type strain. B. noackiue type strain NSW brennerae (bren’ner.ae. M. L. fem. gen. n. brennerue of Bren- 11 (= ATCC 51607 = DSM 9401 = serial no. 170), an EG59- ner, in honor of Frances W. Hickman-Brenner, an American like strain, was isolated from a snail captured in Sydney, New microbiologist, for her contributions to the study of many gen- South Wales, Australia. Good growth occurs at 30 to 36°C. era of the Enterobucteriuceue [4, 9, 10, 161, including the de- Strain NSW llT colonies are circular, convex, greyish, and scription of the genus Kluyveru [ll]) is referred to as DNA smooth on nutrient agar and blood agar, 1 to 2 mm in diameter hybridization group 4 in this study. Biochemical characteristics after 1 day, and 2 to 3 mm in diameter after 2 days. Weak are shown in Table 2. Tests that can be used to differentiate B. growth occurs at 42°C. The type strain has all of the charac- brennerue from other Buttiuuxella species are the arabinose, teristics given for the genus in Table 7 and for the species in arginine dihydrolase, fucose, myo-inositol, 5-ketogluconate, ly- Table 2. sine decarboqlase, and malonate tests. Isolated from mollusks. Description of Buttiauxella warmboldiae sp. nov. Buttiuuxella Description of the type strain. B. brennerue type strain S1/ wurmboldiae (warm’bold.i.ae. M. L. fern. gen. wurmboldiue, of 6-571 (= ATCC 51605 = DSM 9396 = serial no. 145) was Warmbold, in honor of Sabine Warmbold, who isolated most isolated from a snail sampled in Braunschweig, Germany. Its strains of the new Buttiauxellu species at the Staatliches Mediz- colonies are circular, convex, greyish, and smooth on nutrient inaluntersuchungsamt Braunschweig) was referred to as DNA agar and blood agar. They are 1 to 2 mm in diameter after 1 hybridization group 7 in this study. Biochemical characteristics day and 2 to 3 mm in diameter after 2 days. Good growth are shown in Table 2. myo-Inositol is utilized and acid is pro- occurs at 30 to 36”C, and no growth occurs at 42°C. The type duced by freshly isolated strains for many months, in contrast strain has all of the characteristics given for the genus in Table to all other species belonging to the genera Buttiuuxella and 7 and for the species in Table 2. Kluyveru. However, these characteristics are lost after some Description of Buttiauxella izardii sp. nov. Buttiuuxellu izurdii years of storage; therefore, they are not constitutive. Other (iz.ard’i.i. M. L. masc. gen. n. izurdii, of Izard, in honor of biochemical tests that are useful in differentiating B. wurm- Daniel Izard, a French microbiologist, for his contribution to boldiue from other Buttiuuxellu species are the arabinose, ar- the study of the genus Buttiuuxellu [13, 141) was referred to as ginine dihydrolase, citrate, fucose, KCN, malonate, maltitol, DNA hybridization group 5 in this study. Biochemical charac- ornithine decarboxylase, and palatinose tests. As Table 3 teristics are shown in Table 2. No single biochemical charac- shows, this species is the species that is the most distant from teristic differentiates B. izurdii from B. ugrestis, but a combina- all other Buttiuuxellu species and has the highest phenotypic VOL. 46, 1996 NEW BUTTIAUXELLA AND KL.uM/ERA SPECIES 63

probability distance and the lowest level of DNA-DNA relat- 2. Altwegg, M., J. Zollinger-Iten, and A. von Graevenitz. 1986. Differentiation edness. Isolated from snails. of Kluyveru cryocrescens from Kluyvera ascorbutu by irgasan susceptibility Description of the type strain. B. wamboldiae type strain testing. Ann. Microbiol. (Inst. Pasteur) 137A 159-168. 3. Brenner, D. J., G. R. Fanning, A. V. Rake, and K. E. Johnson. 1969. Batch = = NSW 326 (= ATCC 51608 DSM 9404 serial no. 182), an procedure for thermal elution of DNA from hydroxyapatite. Anal. Biochem. EG59-like strain, was isolated from a snail sampled in Sydney, 2k447-459. New South Wales, Australia. Good growth occurs at 30 to 4. Brenner, D. J., J. J. Farmer 111, G. R. Fanning, A. G. Steigenvalt, P. Klykken, 36°C. Strain NSW 326T colonies are circular, convex, greyish, H. G. Wathen, F. W. Hickman, and W. H. Ewing. 1978. Deoxyribonucleic and smooth on nutrient agar and blood agar, 1 to 2 mm in acid relatedness of Proteus and Providenciu species. Int. J. Syst. Bacteriol. 28 diameter after 1 day, and 2 to 3 mm in diameter after 2 days. 269-282. Moderate growth occurs at 42°C. The type strain has all of the 5. Brenner, D. J., P. A. D. Grimont, A. G. Steigerwalt, G. R. Fanning, E. Ageron, and C. F. Riddle. 1993. Classification of citrobacteria by DNA characteristics given for the genus in Table 7 and for the hybridization: designation of Citrobucterfameri sp. nov., Citrobucteryoungue species in Table 2. sp. nov., Citrobucter braukii sp. nov., Citrobacter werkmanii sp. nov., Citro- Description of Kluyvera cochleae sp. nov. Kluyvera cochleae hacter sedlakii sp. nov., and three unnamed Citrobucter genomospecies. Int. J. (coch’le.ae. L. fem. gen. n. cochleae, of a snail) was referred to Syst. Bacteriol. 43:645-658. as DNA hybridization group 8 in this study. Generally less 6. Brenner, D. J., A. C. McWhorter, J. K. Leete-Knutson, and A. G. Steigenvalt. metabolically active than other Kluyvera species; e.g., the ar- 1982. Escherichiu vulneris: a new species of Enterobactetiuceae associated with human wounds. J. Clin. Microbiol. 151133-1 140. abinose, m-coumarate, 3-hydroxyphenylacetate, lactulose, and 7. Dollberg, S., A. Gandacu, and A. Klar. 1990. Acute pyelonephritis due to a 3-phenylpropionate reactions are negative. Negative for in- Kluyvera species in a child. Eur. J. Clin. Microbiol. Infect. Dis. 9281-283. dole production and lysine decarboxylase activity. The negative 8. Ewing, W. H. 1986. Edwards and Ewing’s identification of Enterobacteri- methyl red reaction and positive Voges-Proskauer reaction aceae, 4th ed. Elsevier, New York. distinguish K. cochleae from all Buttiamella species and all 9. Farmer, J. J., 111, M. A. Asbury, F. W. Hickman, D. J. Brenner, and the other Kluyvera species. Other characteristics that differentiate Enterobacteriaceae Study Group. 1980. Enterobacter sukuzakii: a new species of “Enterobacteriaceue” isolated from clinical specimens. Int. J. Syst. Bacte- K. cochleae from other Kluyvera species, as well as Buttiauxella rial. 30569-584. species, are shown in Table 2. K. cochleae exhibits the largest 10. Farmer, J. J., 111, B. R. Davis, F. W. Hickman-Brenner, A. McWhorter, G. P. probabilistic distance to other Kluyvera species (Tables 3 and 4. Huntley-Carter, M. A. Asbury, C. Riddle, H. G. Wathen-Grady, C. Elias, Isolated from mollusks. G. R. Fanning, A. G. Steigerwalt, C. M. O’Hara, G. K. Morris, P. B. Smith, Description of the type strain. K. cochleae type strain S and D. J. Brenner. 1985. Biochemical identification of new species and 3/1-49 (= ATCC 51609 = DSM 9406 = serial no. 185) was biogroups of Enterobucteriaceae isolated from clinical specimens. J. Clin. isolated from a snail sampled in Braunschweig, Germany. Its Microbiol. 21:46-76. 11. Farmer, J. J., 111, G. R. Fanning, G. P. Huntley-Carter, B. Holmes, F. W. colonies are circular, convex, greyish, and smooth on nutrient Hickman, C. Richard, and D. J. Brenner. 1981. Kluyvera, a new (redefined) agar and blood agar. They are 1 to 2 mm in diameter after 1 genus in the family Enterobacteriaceae: identification of Kluyveru uscorbutu day and 2 to 3 mm in diameter after 2 days at 30 to 36°C. sp. nov. and Kluyvera cryocrescens sp. nov. in clinical specimens. J. Clin. Moderate growth occurs at 42°C. The type strain has all of the Microbiol. 13:919-933. characteristics given for the genus in Table 7 and for the 12. Farmer, J. J., 111, and M. T. Kelly. 1991. Enterobacteriaceae, p. 360-383. In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. species in Table 2. Shadomy (ed.), Manual of clinical microbiology, 5th ed. American Society Description of Kluyvera georgiana sp. nov. Kluyvera georgiana for Microbiology, Washington, D.C. (georg’i.a.na. M. L. fem. adj. georgiana, of Georgia, where im- 13. Ferragut, C., D. Izard, F. Gavini, B. Lefebvre, and H. Leclerc. 1981. Butti- portant work on Buttiamella and Kluyvera was done) was pre- auxella, a new genus of the family Enterobucteriaceae. Zentralbl. Bakteriol. viously called Kluyvera species group 3 (11) and in this study Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe C 2:3344. was referred to as DNA hybridization group 9. The 3-phenyl- 14. Gavini, F., D. Izard, C. Ferragut, J. J. Farmer 111, and H. Leclerc. 1983. propionate and m-coumarate reactions differentiate K. georgi- Separation of Kluyvera and Buttiauxella by biochemical and nucleic acid methods. Int. J. Syst. Bacteriol. 33:880-882. ana from K. cochleae. A combination of tests is necessary to 15. Gavini, F., B. Lefebvre, and H. Leclerc. 1976. Positions taxonomiques differentiate K. georgiana from K. ascorbata and K. cryocrescens. d’enterobacteries H,S-par rapport au genre Citrobacter. Ann. Microbiol. Isolated from human sputum and throat cultures. (Inst. Pasteur) 127A275-295. Description of the type strain. The type strain of K. georgi- 16. Hickman-Brenner, F. W., G. P. Huntley-Carter, Y. Saitoh, A. G. Steigerwalt, ana is strain CDC 2891-76 (= ATCC 51603 = DSM 9409 = J. J. Farmer 111, and D. J. Brenner. 1984. Moellerella wisconsensis, a new serial no. 189). It was isolated from human sputum (11). Good genus and species of Enterobucteriaceae found in human stool spccimens. J. Clin. Microbiol. 19460-463. growth occurs at 30 to 42°C. Strain CDC 2891-76T colonies are 17. Luttrell, R. E., G. A. Rannick, J. L. Soto-Hernandez, and A. Verghese. 1988. circular, convex, greyish, and smooth on nutrient agar and blood Kluyveru species soft tissue infection: case report and review. J. Clin. Micro- agar, 1 to 2 mm in diameter after 1 day, and 2 to 3 mm in biol. 26:2650-265 1. diameter after 2 days. The type strain has all of the characteris- 18. Vkron, M. 1975. Nutrition et taxonomie des enterobacteries. I. Methode tics given for the genus in Table 7 and for the species in Table 2. d’ktude des auxanogrammes. Ann. Microbiol. (Inst. Pasteur) 126A267-274. 19. Wayne, L. G., D. J. Brenner, R. R. Colwell, P. A. D. Grimont, 0. Kandler, REFERENCES M. I. Krichevsky, L. H. Moore, W. E. C. Moore, R. G. E. Murray, E. 1. Altwegg, M., A. von Graevenitz, and J. Zollinger-Iten. 1987. Medium and Stackebrandt, M. P. Starr, and H. G. Triiper. 1987. Report of the Ad Hoc temperature dependence of decarboxylase reactions in Aeromonas spp. Curr. Committee on Reconciliation of Approaches to Bacterial Systematics. Int. J. Microbiol. 151-4. Syst. Bacteriol. 37:463-464.