INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1991, p. 88-103 Vol. 41, No. 1 0020-7713/91/010088-16$02.00/0 Copyright 0 1991, International Union of Microbiological Societies

Revision of Campylobacter, Helicobacter, and Wolinella Taxonomy: Emendation of Generic Descriptions and Proposal of Arcobacter gen. nov.

P. VANDAMME,l* E. FALSEN,2 R. ROSSAU,’t B. HOSTE,l P. SEGERS,l R. TYTGAT,l AND J. DE LEY’ Laboratorium voor Microbiologie en Microbiele Genetica, Rijksuniversiteit Gent, B-9000 Ghent, Belgium,’ and Culture Collection, Department of Clinical Bacteriology, University of Goteborg, S-413 46 Goteborg, Sweden2

Hybridization experiments were carried out between DNAs from more than 70 strains of Campylobacter spp. and related taxa and either 3H-labeled 23s rRNAs from reference strains belonging to Campylobacter fetus, Campylobacter concisus, Campylobacter sputorum, Campylobacter coli, and Campylobacter nitrofigilis, an unnamed Campylobacter sp. strain, and a Wolinella succinogenes strain or 3H- or 14C-labeled23s rRNAs from 13 gram-negative reference strains. An immunotyping analysis of 130 antigens versus 34 antisera of campylobacters and related taxa was also performed. We found that all of the named campylobacters and related taxa belong to the same phylogenetic group, which we name rRNA superfamily VI and which is far removed from the gram-negative bacteria allocated to the five rRNA superfamilies sensu De Ley. There is a high degree of heterogeneity within this rRNA superfamily. Organisms belonging to rRNA superfamily VI should be reclassified in several genera. We propose that the emended genus Campylobacter should be limited to Campylobacter fetus, Campylobacter hy ointestinalis , Campylobacter concisus, Campylobacter m ucosalis , Campylobacter sputorum, Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and “Campylobacter upsaliensis. ” Wolinella curva and Wolinella recta are transferred to the genus Campylobacter as Campylobacter curvus comb. nov. and Campylobacter rectus comb. nov., respectively. Bacteroides gracilis and Bacteroides ureolyticus are generically misnamed and are closely related to the genus Campylobacter. Campylobacter nitrofigilis, Campylobacter cryaerophila, and an unnamed Campylobacter sp. strain constitute a new genus, for which the name Arcobacter is proposed; this genus contains two species, Arcobacter nitrofigilis comb. nov. (type species) and Arcobacter cryaerophilus comb. nov. Wolinellu succinogenes so far is the only species of the genus Wolinella. The genus Helicobacter is also emended; Campylobacter cinaedi and Campylobacter fennelliae are included in this genus as Helicobacter cinaedi comb. nov. and Helicobacter fennelliae comb. nov., respectively. The genus “Flexispira,” with “Flexispira rappini” as the only species, is closely related to the genus Helicobacter. The free-living, sulfur-reducing campylobacters do not belong to any of these genera; they probably constitute a distinct genus within rRNA superfamily VI.

At present, the genus Campylobacter consists of 13 well- Dewhirst (39) found a close relationship between Wolinella defined species (40). Recently, two additional species, Cam- curva, Wolinella recta, Bacteroides gracilis, and Bacteroi- pylobacter pylori and Campylobacter mustelae, were in- des ureolyticus on the one hand and the so-called true cluded in the new genus Helicobacter as Helicobacter pylori campylobacters (i-e., the Campylobacter taxa belonging to and Helicobacter mustelae, respectively (20). The clinical rRNA homology group I of Thompson et al. [58] on the significance of all of these organisms was reviewed recently other. These Wolinella and Bacteroides species have been by Penner (40). A study of the taxonomic structure of the isolated from humans with both oral and nonoral infections genus Campylobacter in which partial 16s rRNA sequence (39). analysis was used revealed that the Campylobacter species It was the aim of this study to include all known campylo- can be divided into three major rRNA homology groups (58). bacters and possible relatives in a single phylogenetic study. The first homology group contains Campylobacterfetus (the Therefore, we selected reference strains of the 15 Campylo- type species of the genus Campylobacter), Campylobacter bacter and Helicobacter species, representative strains of hyoin testin ah , Campylo ba cter sp u torum , Campylo ba cter the saprophytic campylobacters (i.e. , the aspartate-ferment- jejuni, Campylobacter coli, Campylobacter lari (62) , “Cam- ing, free-living Campylobacter species of Laanbroek et al. pylobacter upsaliensis,” Campylobacter concisus, and [27] and Spirillum sp. strain 5175 of Wolfe and Pfennig [65]), Campylobacter mucosalis. The second rRNA homology and strain CLO-3 of Fennel1 et al. (16). In addition to these group contains Campylobacter pylori, Campylobacter fen- Campylobacter strains, we also included representative nelliae, and Campylobacter cinaedi; Wolinella succino- strains of Wolinella succinogenes, Wolinella curva, and genes, an organism found in bovine rumina, also belongs to Wolinella recta, and an unnamed Wolinella strain (57), as this second rRNA homology group. The third rRNA homol- ogy group consists of Campylobacter nitrojigilis and Cam- well as representative strains of Bacteroides gracilis, Bac- pylobacter cryaerophila. Furthermore, in another phyloge- teroides ureolyticus, and “Flexispira rappini,” which is a netic study of the genus Campylobacter, Paster and recently described organism that has been isolated from aborted fetuses (5, 26) and diarrheic stools (1). In order to study the genotypic coherence of these organ- * Corresponding author. isms and their phylogenetic relationships with other gram- t Present address: Innogenetics N. V., Antwerp, Belgium. negative bacteria, we used the DNA-rRNA hybridization

88 VOL.41, 1991 TAXONOMY OF rRNA SUPERFAMILY VI 89

technique of De Ley and De Smedt (11) and the immunotyp- cpmtpg for Campylobacter concisus CCUG 20534; 2,000 ing technique of Falsen (15). This enabled us to study several cpm/pg for Arcobacter nitrofigilis CCUG 12022; 70,000 strains of most taxa, whereas only single representative cpm/pg for CLO strain CCUG 10373; and 29,000 cpm/pg for strains have been included in previous partial 16s rRNA Wolinella succinogenes CCUG 13145T. These specific activ- sequence analyses (29, 39, 42, 58). ities were determined with a Beckman model 3310 Tri-Carb Below, we use the new names Campylobacter curvus, scintillation counter. Carnpylobacter rectus, Arcobacter nitrojigilis, Arcobacter DNA-rRNA hybridization experiments. Fixation of single- cryaerophilus, Helicobacter cinaedi, and Helicobacter fen- stranded DNAs on membrane filters, chemical determina- nelliae for the organisms formerly called Wolinella curva, tion of the amounts of DNAs on the filters, saturation Wolinella recta, Carnpylobacter nitrojigilis, Carnpylobacter hybridization, RNase treatment, and measurement of the cryaerophila, Carnpylobacter cinuedi, and Campylobacter thermostabilities of the hybrids were performed as described fennelliae, respectively. previously (11).We used labeled 23s rRNAs from Cumpylo- (Preliminary immunotyping results have been presented bacter, Arcobacter, and Wolinella strains (Table 2) and a previously [15a, 15bl.) variety of gram-negative reference strains (Table 3). Each DNA-rRNA hybrid was characterized by the following two MATERIALS AND METHODS parameters: (i) the melting temperature of elution [T,(,J, which was the temperature at which 50% of a DNA-rRNA Bacterial strains and growth conditions. All of the strains hybrid was denatured; and (ii) the percentage of rRNA used for DNA-DNA hybridization and DNA-rRNA hybrid- binding, which was a way to measure the amount of labeled ization experiments and the representative strains used for rRNA bound to 100 pg of filter-fixed DNA after RNase the immunotyping analysis are shown in Table 1. Names in treatment under standard conditions. A homologous duplex quotation marks have not been validly published. Below, the was formed between the DNA and the rRNA of the same genus names of all generically misnamed taxa are enclosed in strain; a heterologous hybrid was formed between DNA and brackets. Bacteriological purity was checked by plating and rRNA of different strains. The T,(,, is the most important examining living and Gram-stained cells. For mass cultures, parameter for drawing taxonomic conclusions (11, 13); the cells were grown on petri dishes on blood agar media higher the T,(,, of a heterologous hybrid, the more closely containing 5% (voVvol) horse blood and solidified with 1.8% the two strains are related. The Tm(e) values were used to agar. All Wolinella, Bacteroides, and saprophytic Campylo- calculate the average linkage level between each pair of bacter strains were incubated at 37°C in an anaerobic atmo- rRNA branches by using the unweighted average pair group sphere containing 5% CO,, 10% H2, and 85% N,. All other method (49). The percentage of rRNA binding can be useful strains except the Arcobacter nitrofigilis strains were grown for distinguishing strains with similar T,(,) values. The in a microaerophilic atmosphere containing approximately difference between the T,(,, of a homologous duplex and the 5% 0,, 3.5% CO,, 7.5% H,, and 84% N, at 37°C; the cultures Tmc,)of a heterologous hybrid is called the AT,,,,. of Arcobacter nitrofigilis strains were incubated at 30°C. The DNA-DNA hybridization experiments. The degree of DNA- strains to be used as antigens were incubated in a microaero- DNA binding, expressed as a percentage, was determined philic atmosphere containing approximately 4% C02,7% O,, spectrophotometrically by using the initial renaturation rate 27% N,, and 62% H,. method of De Ley and co-workers (10). Each value given Preparation of high-molecular-weight DNAs. High-molecu- below is the average of the values from at least two hybrid- lar-weight native DNAs were prepared from 0.5 to 2 g (wet ization experiments. DNA binding values of 30% or less weight) of cells by using the method of Marmur (30), purified indicate that there is no significant DNA homology. The total by CsCl gradient centrifugation, and stored at -80°C. DNA concentration was about 39 pg/ml, and the optimal Preparation of radioactively labeled rRNAs. Shake cultures renaturation temperature in 2~ SSC (IxSSC is 0.15 M NaCl of Campylobacter fetus subsp. fetus CCUG 6823AT (T = plus 0.015 M sodium citrate, pH 7) was 64.6"C. type strain), Campylobacter sputorum biovar bubulus DNA base compositions. All of the guanine-plus-cytosine CCUG 11289, and Campylobacter coli CCUG 11283T were (G+ C) values were determined by the thermal denaturation grown in nutrient broth no. 2 (catalog no. CM67; Oxoid Ltd., method and were calculated by using the equation of Mar- Basingstoke, United Kingdom). Shake cultures of Carnpylo- mur and Doty (31), as modified by De Ley (8). bacter concisus CCUG 20534 and Arcobacter nitrofigilis Immunotyping. Preparation of soluble, cell wall-free anti- CCUG 12022 were grown in the same broth medium supple- gens and immunization and immunodiffusion experiments mented with 0.2% (wthol) sodium formate and 0.3% (wthol) were performed as described previously (15, 61). disodium fumarate (for Campylobacter concisus CCUG Phenotypic tests. Phenotypic tests were performed with 20534) or 1% (wthol) NaCl (for Arcobacter nitrofigilis CLO strains CCUG 10373, CCUG 10374, and CCUG 10375 CCUG 12022). Shake cultures of Wolinella succinogenes as described previously (22). CCUG 13145T and Campylobacter-like organism (CLO) strain CCUG 10373 were grown in the medium of Wolin et RESULTS al. (66). All cultures were incubated for 1 to 3 days under the appropriate atmospheric and temperature conditions (see DNA base compositions. Within the species Campylobac- above); 2 mCi of 2,8-C3H]adenine was added to 100 ml of a ter concisus we found a G+C range of 37.9 to 40.4 mol% culture in the early log phase of growth for CLO strain (Table 2), which is similar to the results of Roop et al. (43) CCUG 10373, and 1 to 2 mCi of 5,6-[3H]~racilwas added for but higher than the range of values (34 to 38 mol%) found by the other strains. Labeled rRNAs were prepared and sepa- Tanner et al. (54). All of the other DNA base ratios which we rated into 23s and 16s fractions as described by De Ley and determined (Tables 2 and 3) agree with previously published De Smedt (11). The specific activities of the 23s rRNA data (2, 24, 48, 55, 56). fractions were as follows: 14,000 cpm/pg for Campylobacter DNA-rRNA hybridization experiments. Table 3 shows the fetus subsp. fetus CCUG 6823AT; 115,000 cpdpg for Cam- results of the DNA-rRN A hybridizations between DNAs pylobacter sputorum biovar bubulus CCUG 11289; 65,000 from Campylobacter and Arcobacter strains and radioac- TABLE 1. Strains used Strain” Other designations“ Received from“: Source Organisms belonging to rRNA cluster I Campylobacter fetus subsp. fetus CCUG LMG 6442T, N%TC 10842T NCTC Brain of sheep fetus 6823AT C. fetus subsp. fetus ATCC 33246 Mannheim Joint aspirate C. fetus subsp. fetus NIDO 7572 LMG 6569, CCUG 17693 Dekey ser Calf fetus C. fetus subsp. fetus NIDO 212514 LMG 6571, CCUG 17694 Deke y ser Genitals of a bull C. fetus subsp. venerealis CCUG 538T LMG 6443T, NCTC 10354T NCTC Vaginal mucus of a heifer C. fetus subsp. venerealis NIDO 483 LMG 6570, CCUG 7477 Deke y ser Bovine C. fetus B5 Dekey ser C. hyointestinalis CCUG 14169T LMG 7817T, Gebhart 80-4577-4T Gebhart Porcine intestine C. hyointestinalis CCUG 14916 LMG 7538, C 269 Ursing Bovine feces C. hyointestinalis ADRI 1047 LMG 8634, CCUG 24181 Garcia Feces of a diarrheic beef calf C. hyointestinalis CCUG 20823 LMG 8216, CDC D2189 Patton Human stool C. concisus CCUG 13144T LMG 77!BT, FDC 484T Tanner Gingival sulcus C. concisus CCUG 20534 LMG 7789, NCTC 11486 NCTC Periodontal pocket C. concisus CCUG 17580 LMG 7545 Tornqvist Diarrheic feces of 2-yr-old child C. concisus CCUG 18688 LMG 7963 Jansson Feces from woman suffering from fever and diarrhea C. concisus CCUG 19219 LMG 7964, Goodwin 13961 Goodwin Esophagus biopsy specimen C. miicosalis CCUG 6822T LMG 6448T, NCTC llOOOT NCTC Porcine small intestine C. mucosalis CCUG 10771 LMG 7794, NCTC 11001 NCTC Porcine intestine C. mucosalis FS 921177 LMG 8499, CCUG 23201 Lawson Colon of pig with proliferative enteropathy CLO strain CCUG 20705 LMG 7974, Bolton A4 Bolton Porcine intestine Campylobacter sputorum biovar sputo- LMG 7795T, VPI S-17T Holdeman Human mouth rum CCUG 9728T C. sputorum biovar bubulus CCUG 11289 LMG 6447, CIP 53103 CIP Bull sperm C. sputorum biovar bubulus CCUG 886 NCTC 10355 NCTC Semen of normal bull C. sputorum biovar bubulus ATCC 33491 Mannheim C. sputorum biovar fecalis CCUG 12015 LMG 6617, PC 363 Karmali Ovine feces C. sputorum biovar fecalis CCUG 12017 LMG 6618, PC 365 Karmali Ovine feces C. sputorum biovar fecalis CCUG 17761 LMG 8531, NCTC 11415 NCTC Ovine feces C. sputorum biovar fecalis RB6t2 LMG 6728, CCUG 17695B Dekey ser Ovine feces C. cofi CCUG 11283T LMG 6440T, CIP 7080T CIP Porcine feces C. coli CCUG 8169 Human C. coli CCUG 10369 LMG 7535, Skirrow 4620178 Skirrow Porcine placenta C. jejuni subsp. jejuni ATCC 33250 Mannheim Human blood C. jejuni subsp. jejuni CCUG 14914 LMG 7534 Ursing Canine feces C. jejuni subsp. jejuni CCUG 6824 LMG 8553, NCTC 11168 NCTC Human feces C.jejuni subsp. jejuni RV4 LMG 6629, CCUG 17696 Dekeyser Human C. jejuni subsp. jejuni CCUG 10370 LMG 6446 Skirrow C. jejuni subsp. jejuni M2 Mannheim C. jejuni subsp. jejuni JJ91 Mannheim C. jejuni subsp. doylei CCUG 18265 LMG 7790, NCTC 11847 NCTC Human gastric biopsy C. lari NCTC 11352T LMG 8846T, CCUG 23947T NCTC Cloaca1 swab of a herring gull C. lari CCUG 12774 LMG 7607, Skirrow 175182 Skirrow Child, feces C. lari CCUG 18267 LMG 7791, NCTC 11845 NCTC River water “C. upsaliensis” CCUG 14913 LMG 8850, NCTC 11541 Ursing Canine feces “C. upsaliensis” B523 LMG 8852, CCUG 24191 Goossens Human feces “C. upsaliensis” E282 LMG 7917 Goossens Human feces “C. upsaliensis” CCUG 20818 LMG 7915, CDC D533 Patton Human feces C. curvus CCUG 13146T LMG 7609T, VPI 9584T Tanner Human alveolar abscess C. rectus CCUG 20446T NCTC 11489T, FDC 371T NCTC Human periodontitis C. rectus CCUG 11640 LMG 7611, D13a-g, VPI 10278B Sundqvist Human dental root canal [Wolinella]sp. strain CCUG 11641 LMG 8543, VPI 10279 Sundqvist Human dental root canal [Bacteroides]gracilis FDC 404 LMG 7616, CCUG 22762 Tanner Gingival crevice [Bacteroides]ureolyticus CCUG 7319T LMG 6451T, NCTC 10941T NCTC Amniotic fluid [Bacteroides]ureolyticus CCUG 9596 Our own isolate Wound, penis [Bacteroides]ureolyticus CCUG 9510D Our own isolate Wound, penis Organisms belonging to rRNA cluster I1 Arcobacter nitrofgilis CCUG 15893T LMG 7604T, CCUG 15892T, CI’ McClung Roots of Spartina alternifora A. nitrofgilis CCUG 12022 LMG 7547, PC 371 Karmali Roots or root-associated sediment of Spartina alternijlora A. cryaerophilus CCUG 17801T LMG 7536T, Neill A1691BT Neill Aborted bovine fetus A. cryaerophilus CCUG 17805 LMG 7537, Neill B1056lP Neill Aborted ovine fetus A. cryaerophilus CCUG 12018 LMG 6622, PC 367 Karmali Kidney of an aborted porcine fetus A. cryaerophilus CCUG 12019 LMG 9065, Neill 02797 Karmali Placenta of aborted ovine fetus CLO strain CCUG 10373 LMG 6620, Skirrow 996/79 Skirrow Human blood CLO strain CCUG 10374 LMG 6621, Skirrow 449180 Skirrow Feces of lamb with diarrhea CLO strain CCUG 10375 LMG 8538, Skirrow 1018179 Skirrow Bovine Continued on following page

90 VOL. 41, 1991 TAXONOMY OF rRNA SUPERFAMILY VH 91

TABLE 1-Continued

Strain“ Other designations“ Received from”: Source Organisms belonging to rRNA cluster 111 Helicobacter cinaedi CCUG 18818T LMG 7S43T, Fennell 165T Fennel1 Rectal swab of a homosexual male H. cinaedi CCUG 15432 LMG 8559 Claesson Blood of 42-yr-old female H. cinaedi CCUG 17733 LMG 8558 Our own isolate Feces of 1-yr-old female H. fennelliae CCUG 18820T LMG 7546T, Fennell 231T Fennell Rectal swab of a homosexual male H. pylori CCUG 17874T LMG 7S39=, KO0456591 Goodwin Endoscopic biopsy specimen H. pylori CCUG 15816 LMG 8773, CT1 Mars hall Duodenum H. pylori CCUG 19106 LMG 8775, Pylo 10 Megrau d Gastric mucosa H. mustelae CCUG 23652 LMG 8776, LMG 8928, NCTC NCTC Ferret gastric mucosa 12031 “Flexispira rappini” ATCC 43879 LMG 8458, LMG 8738, CCUG ATCC Patient with gastroenteritis 23435 “F. ruppini” ATCC 43880 LMG 8457 ATCC Patient with gastroenteritis Wolinella succinogenes CCUG 13145T LMG 7608T, DSM 1740‘ CCUG Bovine rumen fluid W. succinogrnes DSM 1740T LMG 7466T, CCUG 13145= DSM Bovine rumen fluid CLO-3 strain CCUG 14564 LMG 7792, Skirrow 912/79 Skirrow Rectal swab of a homosexual male Other campylobacters and reference strains CLO strain CCUG 13942 LMG 7793, DSM 806 DSM Anaerobic sludge [Spirillurn] sp. strain 5175 LMG 8192 Pfennig Actinobacillus lignieresii NCTC 4189T Mannheim Bovine lesion Agrobacterium tumefuciens ICPB TTlll LMG 196 ICPB Aquaspirillum aqmticxm ATCC 11330T LMG 2370‘ ATCC Freshwater Aquaspirillum serpens ATCC 12638T LMG 4343T ATCC Arthrobucter oxydans CBRI 21010T LMG 3816T CBRI Air Bacteroides coagulans CCUG 10974T LMG 8206T, ATCC 29798’ ATCC Perineum scar Bacteroides fragih NCTC 9345 Mannheim Bacteroides orulis ATCC 33269= Mannheim Human periodontal pocket Bacteroides splanchnicus ATCC 29572= LMG 8202T ATCC Abdominal abscess Brucella ubortus ATCC 23448T Mannheim Bovine Brucella melitensis NCTC 10094T Mannheim Cardiobucterium hominis ATCC 15826T Mann heim Human blood Cytophaga johnsnnue ATCC 1706IT LMG 1340T NCIB Soil Escherichia coli B LMG 2093 Fusobacterium nucleatum ATCC 25586= Mannheim Cervicofacial lesion Gardnerella vaginalis ATCC 140BT LMG 7832’r ATCC Human vaginal secretions Haemophilus influenzae NCTC 8143T Mannheim Janthinobacterium lividum NCTC 9796T LMG 2892” Sneath Soil Legionella pneumophila ATCC 33153 Mannheim Human lung Leptotrichia buccalis NCTC 10249T Mannheim Supragingival calculus Moraxella lacunata NCTC 7911 LMG 1009, ATCC 17952 NCTC Neisseria Pavescens ATCC 13120T LMG 5297=, CCUG 34ST ATCC Human spinal fluid Oceanospirillurn pusillum IF0 13613T LMG 5308= IF0 Mussel Oligella urethralis CCUG 994 LMG 5304 Bevre Pseudomonas fluorescens ATCC 13525T LMG 1794T MMCA Prefilter waterworks tanks Streptobacillus moniliformis NCTC Mannheim Human case of rat bite fever 10651T Tissierella praeacuta ATCC 25539T LMG 8203T ATCC Gangrene Unidentified strain CCUG 10372 LMG 7819, Skirrow 912/79 Skirrow Equine feces ’ ATCC, American Type Culture Collection, Rockville, Md.; CBRI, Cell Biology Research Institute, Department of Agriculture, Ottawa, Canada; CCUG, Culture Collection of the University of Goteborg, Department of Clinical Bacteriology, University of Goteborg, Goteborg, Sweden; CIP, Collection bactkrienne de I’ Institut Pasteur, Pans, France; DSM, Deutsche Sammlung von Mikroorgmismen, Braunschweig, Federal Republic of Germany; ICPB, International Collection of Phytopathogenic Bacteria, Department of Bacteriology, University of California, Irvine; IFO, Institute for Fermentation, Osaka, Japan; LMG, Culture Collection, Laboratorium voor Microbiologie, University of Ghent, Ghent, Belgium; MMCA, Medical Microbiology Culture Collection, Aarhus, Denmark; NICB, National Collection of Industrial Bacteria, NCIMB Ltd., Torry Research Station, Aberdeen, United Kingdom; NCTC. National Collection of Type Cultures, Central Public Health Laboratory Services, London, United Kingdom; Bolton, F. Bolton, Public Health Laboratory Service, Preston Infirmary, Preston, United Kingdom; Bgvre, K. Bgvre, Kaptein W. Wilhelmsen og Frues Bakteriologiske Institutt, University of Oslo, Oslo, Norway; Claesson, B. Claesson, Laboratory for Clinical Bacteriology, Skovde, Sweden; Dekeyser. P. Dekeyser, Nationaal Instituut voor Diergeneeskundig Onderzoek (NIDO), Brussels, Belgium; Fennell, C. L. Fennell, Department of Medicine, Harborview Medical Center, Seattle, Wash. ; Garcia, M. Garcia, Animal Diseases Research Institute (ADRI), Nepean, Ontario, Canada; Gebhart, C. Gebhart, Department of Veterinary Diagnostic Investigation, College of Veterinary Medicine, University of Minnesota, St. Paul; Goodwin, C. S. Goodwin, Department of Microbiology, Royal Perth Hospital, Perth, Western Australia, Australia; Goossens, H. Goossens, World Health Organization Collaborating Center for Enteric Campylobrzcfer,St. Pieters University Hospital, Brussels, Belgium; Holdeman, L. V. Holdeman, Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University (VPI), Blacksburg; Jansson, G. Jansson, Laboratory of Clinical Bacteriology, Regionsjukhuset, Orebro, Sweden; Karmali, M. A. Karmali, Hospital for Sick Children and University of Toronto, Toronto, Ontario, Canada; Lawson, G. Lawson, Department of Veterinary Pathology, University of Edinburgh, Edinburgh, United Kingdom; Mannheim, W. Mannheim, Zentrum fur Hygiene und medizinische Mikrobiologie, Klinikum der Philipps Universitbit, Marburg-Lahn, Federal Republic of Germany; Marshall, B. J. Marshall, Fremantle, Western Australia; McClung, C. McClung, Department of Microbiology and Public Health, Michigan State University, East Lansing; Megraud, F. Mkgraud, H6pital des Enfants, Centre Hospitalier .Regional, Bordeaux, France; Neill. S. D. Neill, Veterinary Research Laboratories, Stormont, Belfast, Northern Ireland; Patton, C. Patton, Centers for Disease Control (CDC), Atlanta. Ga. ; Pfennig, N. Pfennig, Universitat , Konstanz, Federal Republic of Germany; Skirrow, M. B. Skirrow, Public Health Laboratory, Worcester, United Kingdom; Sneath, P. Sneath, Department of Microbiology, Leicester University, Leicester, United Kingdom; Sunddqvist, G. Sundqvist, Department of Endodontics, Faculty of Odontology, University of Ume5, Umei, Sweden; Tanner, A. Tanner, Forsyth Dental Center (FDC), Boston, Mass.; Tornqvist, E. Tornqvist, Laboratory of Clinical Bacteriology, Regionsjukhuset, Orebro, Sweden; Ursing, J. Ursing, Department of Medical Microbiology, University of Lund, Malmo General Hospital. Malmo, Sweden. tl and t2 refer to different colony types. TABLE 2. DNA base compositions and parameters of the DNA-rRNA hybrids formed by using labeled 23s rRNAs from Campylobacter, Arcobacter, and Wolinella strains WN Hybridized with rRNA from:

Campylobacter CamW1obacter campyl0- Arcobacter Wolinella Camwlobacter sputorum G+C fetus subsp. concisus bacter coli nitrofigilis Source of DNA content CCUG CCUG 20534 b$~~~~~~$CCUG 11283T CCUG 12022 CCUG 10373 CCUG 1314ST U (mol%) 6823AT

Organisms belonging to rRNA cluster I Campylobacter fetus subsp. fetus CCUG 6823AT 33.9 76.6 0.23 71.7 0.22 72.1 0.16 69.3 0.17 C. fetus subsp fetus ATCC 33246 33.7 76.4 0.17 71.8 0.17 71.3 0.13 70.7 0.16 66.6 0.11 64.9 0.13 C. fetus subsp. fetus NIDO 212514 34.5 76.9 0.22 71.7 0.22 70.3 0.16 C. fetus subsp. fetus NIDO 7572 34.3 76.5 0.28 71.5 0.17 C. fetus subsp. venerealis CCUG 53ST 33.2 76.6 0.18 71.3 0.16 71.1 0.15 C. fetus supsp. venerealis NIDO 483 33.9 76.3 0.16 64.1 0.09 C. hyointestinalis CCUG 14169T 35.1 73.5 0.12 67.9 0.12 C. hyointestinalis CCUG 24181 33.6 74.8 0.15 63.4 0.08 C. hyointestinalis CCUG 14916 35.2 76.6 0.17 71.8 0.21 71.7 0.17 C. hyointestinalis CCUG 20823 74.7 0.15 70.3 0.12 C. concisus CCUG 13144T 37.9 70.9 0.13 76.0 0.18 70.7 0.12 C. concisus CCUG 20534 38.4 72.3 0.13 77.3 0.20 71.5 0.13 69.2 0.15 65.8 0.26 C. concisus CCUG 17580 38.6 71.8 0.14 76.3 0.19 71.4 0.14 69.1 0.14 C. concisus CCUG 18688 39.5 75.2 0.12 C. concisus CCUG 19219 40.4 75.5 0.25 63.7 0.13 C. mucosalis CCUG 6822T 37.1 72.3 0.11 74.9 0.15 71.2 0.11 70.2 0.13 68.2 0.09 C. mucosalis CCUG 10771 36.1 73.4 0.14 71.6 0.10 C. mucosalis FS 921177 37.4 73.5 0.16 70.4 0.12 CLO strain CCUG 20705 34.2 71.4 0.11 72.3 0.15 70.1 0.11 C. sputorum biovar sputorum CCUG 972ST 30.8 70.7 0.14 76.9 0.20 C. sputorum biovar bubulus CCUG 11289 29.9 70.9 0.10 76.5 0.12 69.7 0.09 C. sputorum biovar bubulus ATCC 33491 29.8 70.2 0.13 76.5 0.19 70.0 0.16 C. sputorum biovar fecalis CCUG 12015 30.5 70.7 0.16 76.9 0.23 70.4 0.19 C. sputorum biovar fecalis CCUG 12017 31.8 71.5 0.14 71.8 0.19 77.1 0.21 63.6 0.35 C. sputorum biovar fecalis CCUG 17695B 31.8 76.1 0.16 64.6 0.09 61.8 0.09 C. coli CCUG 11283= 30.8 70.6 0.12 71.8 0.14 77.1 0.23 63.8 0.29 C. coli CCUG 10369 32.5 69.9 0.11 76.2 0.16 62.5 0.07 C. jejuni subsp. jejuni ATCC 33250 30.0 69.4 0.15 71.5 0.17 76.6 0.25 C. jejuni subsp. jejuni CCUG 14914 32.6 70.7 0.15 70.7 0.15 76.3 0.19 C. jejuni subsp. jejuni RV4 30.9 69.5 0.10 70.3 0.12 76.3 0.18 C. jejuni subsp. jejuni CCUG 10370 30.1 70.0 0.14 70.9 0.15 75.8 0.20 C. jejuni subsp. doylei CCUG 18265 30.5 67.1 0.10 77.1 0.16 C. lari NCTC 11352T 30.4 68.5 0.10 73.1 0.19 C. lari CCUG 12774 31.8 70.0 0.15 74.7 0.20 C. lari CCUG 18267 30.5 75.1 0.18 “C. upsaliensis” CCUG 14913” 33.5 69.2 0.15 69.4 0.16 73.3 0.17

“C. upsaliensis” B523 32.8 69.1 0.11 73.6 0.15 v) “C. upsaliensis” E282 32.8 68.0 0.11 68.3 0.11 72.5 0.14 v1 “C. upsaliensis” CCUG 20818 35.4 66.9 0.12 72.0 0.17 C. cuwus CCUG 13146T 45.1 71.0 0.10 69.4 0.13 70.7 0.10 67.2 0.09 62.2 0.06 p C. rectus CCUG 11640 45.4 71.5 0.10 71.6 0.13 71.4 0.11 67.7 0.11 62.9 0.10 [Wolinella] sp. strain CCUG 11641 45.7 72.2 0.13 68.2 0.10 62.7 0.08 3 E [Bacteroides]gracilis FDC 404 69.6 0.10 67.1 0.10 62.4 0.18 0 [Bacteroides] ureolyticus CCUG 7319T 29.5 68.9 0.15 70.0 0.19 67.4 0.19 r Organisms belonging to rRNA cluster I1 Arcobacter nitrofigilis CCUG 15893T 29.0 64.9 0.07 65.2 0.06 76.3 0.11 72.7 0.14 64.5 0.07 A. nitrofigdis CCUG 12022 28.5 66.6 0.09 65.7 0.09 76.7 0.17 73.3 0.16 64.2 0.08 A. cryaerophilus CCUG 17801T 28.6 66.2 0.13 65.9 0.12 71.8 0.17 72.6 0.20 A. cryaerophilus CCUG 17805 28.6 68.5 0.11 67.8 0.06 73.2 0.17 74.1 0.18 65.6 0.14 A. cryaerophilus CCUG 12018 28.4 66.8 0.12 67.9 0.15 72.3 0.17 73.4 0.26 CLO strain CCUG 10373 28.0 66.0 0.15 73.8 0.20 77.4 0.34 65.7 0.18 CLO strain CCUG 10374 29.2 70.1 0.17 71.9 0.18 CLO strain CCUG 10375 29.4 72.4 0.18 71.4 0.19 Organisms belonging to rRNA cluster I11 Helicobacter cinaedi CCUG 188BT 36.1 64.4 0.04 64.2 0.12 71.2 0.05 H. cinaedi CCUG 15432 37.3 64.0 0.06 71.9 0.08 H. cinaedi CCUG 17733 38.3 63.3 0.06 71.2 0.06 H. fennelliae CCUG 18820T 34.9 62.8 0.07 63.2 0.08 71.1 0.09 H. pylon’ CCUG 17874T 35.6 62.2 0.08 62.7 0.09 68.5 0.11 H. pylon CCUG 19106 36.8 64.1 0.05 63.1 0.05 69.6 0.10 H. mustelae CCUG 23652 42.2 64.4 0.04 64.3 0.04 71.3 0.10 CL03 strain CCUG 14564 47.4 63.5 0.04 69.8 0.05 “Flexispira rappini” ATCC 43879 33.7 63.4 0.05 70.5 0.07 “F. rappini” ATCC 43880 34.3 63.6 0.06 62.4 0.14 71.3 0.08 Wolinella succinogenes CCUG 13145T 47.2 79.1 0.20 W succinogenes DSM 1740T 47.0 64.4 0.12 65.8 0.13 67.0 0.13 78.4 0.18 Other campylobacters and reference strains CLO strain CCUG 13942 41.6’ 65.3 0.06 63.7 0.05 [Spirillum]sp. strain 5175 38.4‘ 65.1 0.06 64.9 0.07 Aquaspinllurn serpens ATCC 1263gT 50.0 54.0 0.09 Bacteroides coagulans CCUG 10974T 36.3 53.6 0.05 Bacteroides fiagilis NCTC 9345 47.3 0.16 Bacteroides oralis ATCC 33269T 52.7 0.04 Bacteroides splanchnicus ATCC 29572T 43.1 52.5 0.03 Brucella melitensis NCTC 10094T 57.9 58.8 0.04 Fusobacten’umnucleaturn ATCC 25586T 52.0 0.05 Gardnerella vaginalis ATCC 1401gT 42.0 50.2 0.03 Haemophilus influenzae NCTC 8143T 38.9 57.8 0.12 Legionella pneurnophila ATCC 33153 37.8 58.7 0.03 Leptotnchia buccalis NCTC 10249T 53.8 0.05 Oceanospirillum pusillurn IF0 13613T 51.0 58.8 0.08 Streptobacillus moniliformis NCTC 10651T 24.2 52.4 0.09 v1 Tissierella praeacuta ATCC 25539T 29.3 51.8 0.09 Unidentified strain CCUG 10372 58.5 55.7 0.10 8 Reference strain of Sandstedt et al. (47). Data from reference 27. 5F Data from reference 65. 2 c,

wW 94 VANDAMME ET AL. INT. J. SYST.BACTERIOL.

TABLE 3. Parameters of DNA-rRNA hybrids between DNAs from Campylobacter and Arcobacter strains and 3H-labeled 23s rRNAs from several gram-negative reference strains

% of rRNA DNA from: Labeled rRNA from: TmW ("C) binding Campylobacter fetus subsp. fetus CCUG 6823AT Cardiobacterium hominis ATCC 15826T 60.1 0.07 C.fetus subsp. fetus NIDO 7572 Cardiobacterium hominis ATCC 15826T 58.1 0.07 C.fetus subsp. fetus NIDO 7572 Cytophaga johnsonae ATCC 17061T 55.3 0.04 C.fetus subsp. fetus NIDO 7572 Oligella urethralis CCUG 994 52.4 0.05 C. fetus subsp. fetus NIDO 7572 Brucella abortus ATCC 2344gT 56.9 0.08 C. feius subsp. .fetus NIDO 7572 Neisseria javescens ATCC 13120T 53.8 0.08 C. fetus subsp. fetus NIDO 212514 Cardiobacterium hominis ATCC 15826* 53.2 0.07 C. fetus subsp. fetus NIDO 212514 Moraxella lacunata ATCC 17952 55.7 0.06 C. fetus subsp. fetus NIDO 212514 Actinobucillus lignieresii NCTC 4189T 57.2 0.05 C. fetus subsp. venerealis CCUG 7477 Cardiobacterium hominis ATCC 15826T 57.4 0.05 C.fetus B5 Janthinobacterium lividum NCTC 9796Ta 55 .O 0.12 C. fetus B5 Arthrobacter oxydans CBRI 21010T 58.0 0.14 C. fetus B5 Escherichiu coli B 59.5 0.08 C. jejuni subsp. jejuni ATCC 33250 Curdiobacterium hominis ATCC 15826T 60.1 0.07 C. jejuni subsp. jejuni M2 Pseudomonas Jluorescens ATCC 13525=' 55.0 0.10 C. jejuni subsp. jejuni M2 Janthinobacterium lividum NCTC 9796T" 57.0 0.10 C.jejuni subsp. jejuni M2 Agrobacterium tumefaciens ICPB TTlll 57.0 0.07 C. jejuni subsp. jejuni M2 Escherichia coli B 58.0 0.06 C. jejuni subsp. jejuni JJ91 Pseudomonas Jluorescens ATCC 13525Tb 54.0 0.07 C. jejuni subsp. jejuni CCUG 10370 Cardiobacterium hominis ATCC 15826T 59.7 0.06 C. sputorum biovar bubulus ATCC 33491 Cardiobacterium hominis ATCC 15826T 58.8 0.06 C. sputorum biovar bubulus ATCC 33491 Aciinobacillus lignieresii NCTC 4189T 57.5 0.04 C. sputorum biovar bubulus ATCC 33491 Neisseria Jlavescens ATCC 13120T 52.2 0.05 Arcobacter nitroJgilis CCUG 12022 Aquaspirillum ayuaticum ATCC 11330T 51.6 0.09

(I Data from reference 12. ' Data from reference 13. tively labeled rRNAs from strains belonging to gram-nega- The following three strains also belong to rRNA cluster I: tive reference taxa. Table 2 shows the results of DNA-rRNA CLO strain CCUG 20705, [Wolinella]sp. strain CCUG 11641 hybridizations between DNAs from campylobacters, related (57), and strain CCUG 18267. The latter strain was received taxa, and gram-negative reference strains and radioactive as a representative of the urease-positive therrnophilic Cam- rRN As from Campyfobacter, Arcobacter, and Wolinelln pylobacter group (3,4). This group of thermophilic campylo- strains. The DNA-rRNA hybridization results are presented bacters was identified as Campylobacter lari (38) by the as a dendrogram based on the T,(,, values of the hybrids in sodium dodecyl sulfate-polyacrylamide gel electrophoresis Fig. 1. method. We performed DNA-DNA hybridizations between The DNA-rRNA hybridizations between DNAs from cam- this strain and Campylobacter lari CCUG 12774 and found a pylobacters and related strains and rRNAs from gram- level of DNA binding of 67% (data not shown). When negative reference strains belonging to rRNA superfamilies I DNA-DNA hybridizations were performed between DNA to (Tables 2 and 3) revealed Tmcc)values between and V 50.2 from strain CCUG 20705 and DNAs from Campylobacter 60.4"C (average, 56.6 2 3.1"C). fetus, Campylobacter hyointestinalis, Campylobacter con- The DNA-rRNA hybridization results show that within rRNA superfamily VI, three major rRNA homology groups, cisus, and Campylobacter mucosalis strains, no significant called rRNA clusters I, 11, and 111, can be differentiated (Fig. DNA binding values were detected (data not shown). 1).rRNA cluster I is linked to rRNA cluster 11 at an average rRNA cluster I1 consists of Arcobacter nitrojigilis (two strains), Arcobacter cryaerophilus (three strains), and CLO Tm(c)of 65.9 5 1.6"C, and rRNA cluster I11 is linked to rRNA clusters I and I1 at an average Tnl(,) of 63.7 2 1.2"C. The strains CCUG 10373, CCUG 10374, and CCUG 10375. structure of these rRNA clusters is shown in Fig. 1. rRNA rRNA cluster 111 consists of Wolinella succinogenes (two cluster I contains Campylobacter fetus subsp. fetus (four strains), Helicobacter pylori (two strains), Helicobacter strains), Campylobacter fetus subsp. venerealis (two mustelae (one strain), Helicobacter cinaedi (three strains), strains), Campylobacter hyointestinalis (four strains), Cam- Helicobacter fennelliae (one strain), "Flexispira rappini" pylobacter concisus (five strains), Campylobacter mucosalis (one strain), and strain CLO-3 of Fennel1 et al. (16). (three strains), Campylobacter sputorum (six strains repre- The free-living campylobacters of Laanbroek et al. (27) senting Campylobacter sputorum biovar sputorum, Cam- and Wolfe and Pfennig (65) do not belong to one of the three pylobacter sputorum biovar bubulus, and Campylobacter major rRNA homology groups. They have a separate posi- sputorum biovar fecalis), Campylobacter coli (two strains), tion on the T,,z(c)dendrogram (Fig. 1) at an average T,(,, of Campylobacterjejuni subsp. jejuni (four strains), Campylo- 643°C (Table 2). bacter jejuni subsp. doylei (one strain), Campylobacter lari DNA-DNA hybridization results. DNA-DNA hybridiza- (two strains), "Campylobacter upsaliensis" (four strains), tions were performed with nine strains belonging to rRNA Campylobacter curvus (one strain), Campylobacter rectus cluster 11 (Fig. 2). Our hybridization results revealed the (one strain), and the generically misnamed organisms [Bac- presence of three DNA homology groups and one separate teroides] gracilis (one strain) and [Bacteroides] ureolyticus strain (CLO strain CCUG 10373). Within the groups, the (one strain). DNA binding values varied from 46 to 100%. Significant VOL.41, 1991 TAXONOMY OF rRNA SUPERFAMILY VI 95

Campylobacter concisus Campylobacter mucosalis ---- CLO strain CCUG 20705 Campylobacter sputorum Campylobacter curvus - I Campylobacter rectus Y rRNA cluster I I [Wolinella] sp. CCUG 11641 I I I -“Campylobacter upsaliensir” I - 7+I Campylobacter [;!ni

I- Campylobacter lari i [Bacteroides] ureolyticus I [Bacteroides] gracilis

Arcobacter nitrofigilis CLO strain CCUG 10373 II.. I --- Arcobacter cryaerophilus strains CCUG 10374 CLO [CCUG 10375

r-- CLO-3 I r - “Flexispira rappini” ~n Wolinella succinogenes

v rRNA superfamily I to V

FIG. 1. Simplified rRNA cistron similarity dendrogram of rRNA superfamily VI. The bars indicate the Tmc,, ranges observed within a species or small group. The dashed lines represent one or more rRNA branches for which no labeled rRNAs are available yet.

degrees of DNA binding were detected between CLO strain biovar fecalis); all Campylobacter sputorum strains gave CCUG 10374 and Arcobacter cryaerophilus strains. similar precipitation values against the three antisera. ITG 4 Immunotyping analysis. Immunizing of rabbits was started contained Campylobacter curvus and Campylobacter rectus in January 1980. We prepared 130 soluble antigens and 34 (subgroups 4a and 4b, respectively). ITG 5 contained all of antisera, representing all of the taxa belonging to rRNA the taxa belonging to the Campylobacter coli rRNA branch, superfamily VI. We prepared antigens from at least the type including Campylobacter coli, Campylobacter jejuni subsp. strain of each taxon. The numbers of strains tested per taxon jejuni, Campylobacter jejuni subsp. doylei, Campylobacter and the average precipitation values versus each antiserum lari, and “Campylobacter upsaliensis,” each of which con- are shown in Table 4. A total of 16 major groups, 6 of which stituted a separate subgroup (subgroups 5a through 5e, could be further subdivided, were delineated (Table 4). respectively). ITGs 6, 7, and 8 each contained a single taxon Significant cross-reactions (values of 13) were observed ([Bacteroides]gracilis, [Bacteroides] ureolyticus, and Arco- only between members of the same rRNA branch. Immuno- bacter nitrofigilis, respectively). Arcobacter cryaerophilus typing group (ITG) 1 contained Campylobacter fetus and and CLO strains CCUG 10373 and CCUG 10374 constituted Campylobacter hyointestinalis. Campylobacterfetus subsp. ITG 9. Arcobacter cryaerophilus and CLO strain CCUG fetus and Campylobacter fetus subsp. venerealis formed 10374 were immunologically similar (subgroup 9a); CLO subgroup la, and Campylobacter hyointestinalis constituted strain CCUG 10373 (subgroup 9b) gave weaker, but still subgroup lb. Campylobacter concisus, Campylobacter mu- clear-cut cross-reactions versus Arcobacter cryaerophilus cosalis, and CLO strain CCUG 20705 constituted ITG 2 and CLO strain CCUG 10374 (Table 4). ITG 10 contained (subgroups 2a through 2c, respectively). ITG 3 contained the only Wolinella succinogenes. ITG 11 consisted of Helico- organisms of the Campylobacter sputorum rRNA branch bacter cinaedi (subgroup 1la) and Helicobacter fennelliae (Campylobacter sputorum biovar sputorum, Campylobacter (subgroup llb). Helicobacter pylori, Helicobacter mustelae, sputorum biovar bubulus, and Campylobacter sputorum “Flexispira rappini,” strain CLO-3, and the Campylobacter TABLE 4. Results of immunotyping analysis

~~ ~ Reaction with antiserum againsta:

No. of Antigen(s) from: strains ITG tested 13 3 8

Camwlobacterfetus subsp. 9 la 8 8 5 0 0 2 10 0 0 000 0 0 00 0 000 0 0 0 0000000000 fern C. fetus subsp. veneredis 5 la 8 7 6 0 0 2 10 0 0 000 0 0 00 0 000 0 0 0 0000000000 C. hyointestinalis 8 lb 5 6 7 0 0 2 2 0 0 0 000 0 0 00 0 000 0 0 0 0000000000 C. concisus 18 2a 0 0 3 7 8 5 6 1 1 0 000 0 0 00 0 012 0 0 0 0000000000 C. mucosalis 11 2b 1 1 3 2 0 8 6 1 0 0 211 0 0 00 0 012 0 0 0 0000000000 CLO strain CCUG 20705 2c 0 0 3 2 0 2 8 2 0 0 102 0 100 0 012 0 0 12010000000 Camwlobacter sputorum 3 0 0 1 0 0 138 8 3 002 0 101 0 0-3 0 0 0 1020000000 biovar sputorum CCUG 9728" C. sputorum biovar bubulus 5 3 1 0 2 0 0 138 7 4 102 0 0 0-1 233 1 0 0 0020000000 C. sputorum biovar fecalis 3 3 0 0 1 0 0 0 2 8 7 3 202 0 0 01 0 122 0 0 1003000000 1 C. curvus CCUG 13146" 4a 0 0 3 1 2 3 3 1 1 0 722 0 100 0 013 0 0 0 0020010000 C. rectus CCUG 20446" 4b 0 0 2 1 0 2 2 2 0 0 782 0 110 0 014 0 0 0 0010000000 C. coli 7 5a 0 1 1 0 0 0 2 0 1 0 2-7 5 4 22 3 022 0 0 0 0020000000 C. jeuni subsp. jejuni 14 5b 0 1 2 0 0 0 2 0 2 0 206 6 4 14 2 122 0 0 0 0011000000 C. jejuni subsp. doylei 3 SC 0 1 2 0 0 0 2 - 2 0 1-6 6 7 23 2 121 0 0 0 10121-0000 C. lari 3 5d 0 1 2 1 1 131 1 0 3-5 2 2 47 2 112 0 0 0 0020000000 "C. upsaliensis" 6 5e 0 0 2 0 0 0 3 0 0 0 306 3 3 14 5 113 0 0 0 0020000000 [Bacteroides]ureolyticus 2 6 0 0 0 0 0 131 0 0 102 0 101 0 884 0 0 0 0100000000 [Bacteroides]gracilis 2 7 0 0 0 0 0 0 0 0 0 0 210 0 0 00 0 017 0 0 0 0000000000 Arcobacter nitrofigilis 3 8 0 0 0 0 0 0 11 0 0 000 0 0 00 0 011 6 0 12000000000 A. cryaerophilus 4 9a 0 0 1 0 0 0 2 0 0 0 --2 0 0 01 0 011 2 6 6 83210-0000 CLO strain CCUG 10373 9b 0 0 0 0 0 0 -0 0 0 --2 0 0 0-0 01-0 0 3 47-00-0000 Wolinella succinogenes 10 0 0 0 0 0 0 0 0 1 0 001 0 100 0 001 0 0 10060000000 CCUG 13145" Helicobacter cinaedi 8 lla 0 0 0 0 0 0 10 0 0 001 0 0 00 0 023 0 0 0 0026320000 H. fennelliae CCUG 18820T llb 0 0 0 0 0 100 0 0 100 0 100 0 002 0 0 0 0023010020 H. pylon 7 12 0 0 1 0 0 0 -0 0 0 000 0 100 0 011 1 0 0 0000016700 H. mustelae 2 13 0 0 0 0 0 0 0 0 0 0 000 0 0 00 0 012 0 0 0 0030032300 "Flexispira rappini" ATCC 14---- 0 0 0 0 0 0 000 0 0 00 0 001 1 0 0 0000060000 43879 CLO-3 strain CCUG 14564 15 0 0 0 0 0 020 0 ---10 - 0- 0 -- 10 0 0 0022100060 CLO strain CCUG 13952 16 102 0 0 020 0 ---2 0 0 0- 3 0-0 3 0 2 00000-0005

a 0, No precipitate; 1 or 2, weak reaction with uncertain interpretation; 3, weak reaction, usually revealing some relatedness; 4 or 5, moderate reaction, revealing relatedness or identity with unsatisfactory antigen or serum; 6, 7, or 8, strong reaction, observed only with closely related strains; -, not performed. Each value is the average of the values obtained from at least two immunodifision analyses. VOL.41, 1991 TAXONOMY OF rRNA SUPERFAMILY VI 97

TABLE 5. Characteristics that differentiate species within the genus Arcobacter

Anaerobic Susceptibility to: growth Growth Urease Nitro- Reduction Hydrolysis Hydrogen Aerobic without in the Colony Swarming Taxon activity activitygenase of nitrites of indoxyl on moist acetate production" Nalidixic Cepha- growth aspartateand presenceof 3.5% media acidb lothinb NaCl furnarate A rcobacter Vd + - - + S S - - + White - nitrojigilis' Arcobacter - - + + - V R + + - Yellow or + cryaerophilus' colorless CLO strains CCUG 10374 - ND ND + - S S + - - Colorless + - - CCUG 10375 - ND ND S S + - - Colorless + CCUG 10373 - ND ND + - S R + - - Yellow + " H,S production was determined in triple sugar iron agar. 30-kg disks. Data from references 32, 34, and 37. +, Reaction is positive for 85% of the strains; -, reaction is negative for 85% of the strains; V, reaction is positive for 15 to 85% of the strains; S, susceptible; R, resistant; ND, not determined.

species of Laanbroek et al. (27) each constituted a separate negative bacteria. These organisms definitely belong to a ITG (ITGs 12 through 16, respectively). separate eubacterial phylum, as suggested by Romaniuk et Phenotypic tests. The results of the phenotypic tests per- al. (42). formed with CLO strains CCUG 10373, CCUG 10374, and Taxonomic structure of the genus Campylobacter and rela- CCUG 10375 are included in Table 5 and in the description of tionships with other bacteria belonging to rRNA superfamily the genus Arcobacter given below. M. In agreement with the results of Paster and Dewhirst (39) and Thompson et al. (58), we found an extremely high level DISCUSSION of phylogenetic heterogeneity within the previously defined genus Campylobacter. This heterogeneity can be compared In previous papers by workers from our laboratory, the with the heterogeneity within the largely generically mis- significance and applications of the DNA-rRNA hybridiza- named genus Pseudomonas (13). Organisms belonging to tion technique have been established (12-14, 45, 46, 63). In different rRNA homology groups of the genus Pseudomonas the last few years, DNA-rRNA hybridization results have have AT,,,, values of up to 10 to WC, values which are been confirmed by rapidly accumulating 16s rRNA sequenc- similar to the AT,,,, values for the organisms belonging to ing data (64). On the basis of T,(,, values, a phylogenetic the three major rRNA homology groups within rRNA super- tree of the major part of the gram-negative bacteria has been family VI (Fig. 1). Cluster analysis of the T,(,) values constructed (9, 13). Within this phylogenetic tree, it is revealed the presence of three major rRNA clusters (Fig. 1). possible to distinguish at least five major groups, called The compositions of these rRNA clusters entirely coincide rRNA superfamilies, which are related only above the family with the compositions of the rRNA homology groups in- level (9). ferred from 16s rRNA sequence analysis (39, 58); only their Preliminary DNA-rRNA hybridization results (60) indi- branching order is reversed. These high AT,,,, values (ap- cated that the genus Campylobacter does not belong to one proximately 10 to WC) undoubtedly reflect relationships at of the five rRNA superfamilies. The purposes of this study or above the genus level (11,13). Therefore, the taxonomy of were (i) to determine the taxonomic position of the genus the former genus Campylobacter and related taxa should be Campylobacter; (ii) to unravel its genotypic structure; and revised. (iii) to determine the phylogenetic relationships among the rRNA cluster I. The rRNA branches within rRNA cluster genera Campylobacter, Wolinella, Helicobacter, and "Flex- I are linked at AT,,,, values of about 6 to 8°C (Fig. 1).Within ispira" and related organisms. Therefore, we prepared each rRNA branch, all of the taxa have significant precipi- seven radioactive labeled rRNAs, more than 70 DNAs, 34 tation values, which reveal immunological similarities within antisera, and 130 antigens from campylobacters and related each rRNA branch (Table 4). Differences in T,(,) values of 6 organisms. to 8°C could point to intergeneric relationships provided Taxonomic position of the genus Campylobacter. The DNA- sufficient phenotypic arguments are available. However, as rRNA hybridizations between Campylobacter strains and Paster and Dewhirst have noted (39), there are at present few gram-negative reference strains belonging to rRNA super- known characteristics which separate the taxa belonging to families I to V (Tables 2 and 3) revealed an average T,(,, rRNA cluster I. It is striking that all thermophilic (or value of 56.6 5 3.1"C. This confirms and extends our thermotolerant), enteropathogenic Campylobacter species preliminary results (60), namely, that the genus Campylo- belong to the Campylobacter coli rRNA branch (Fig. 1).The bacter and its relatives do not belong to one of the five Campylobacter sputorum rRNA branch contains organisms previously described rRNA superfamilies (sensu De Ley [9]) that are commensal for humans and animals, while the within the gram-negative bacterial group, but constitute the Campyfobacter fetus and Campylobacter concisus rRNA core of a sixth rRNA superfamily. The value 56.6"C is the branches contain organisms that are pathogenic or associ- lowest average linking level between two rRNA superfami- ated with diseases, mainly in animals. The former Wolinella lies; it indicates that the genus Campylobacter and related strains (Campylobacter curvus, Campylobacter rectus, and taxa are phylogenetically far removed from the other gram- [ Wolinella] sp. strain CCUG 11641), [Bacteroides] gracilis, 98 VANDAMME ET AL. INT. J. SYST. BACTERIOL. and [Bacteroides] ureolyticus are found in oral cavities in Campylobacter taxa have characteristics that do not corre- higher numbers in patients with periodontal disease than in spond to the characteristics given in the description of the healthy individuals. However, there are so far no phenotypic genus Campylobacter in Bergey ’s Manual of Systematic characteristics that are useful for differentiating the groups Bacteriology (48). Therefore, below we propose an emended of taxa belonging to each rRNA branch. On the contrary, all description of the genus Campylobacter. Campylobacter and former Wolinella strains belonging to Emended description of the genus Campylobacter Sebald rRNA cluster I share a considerable number of phenotypic and Veron 1963. Slender, spirally curved, gram-negative features (see below). Furthermore, they have similar iso- rods that are 0.2 to 0.5 pm wide and 0.5 to 5 pm long. The prenoid quinone compositions (35, 36), which is a taxonom- rods may have one or more spirals and can be as long as 8 ically valuable feature (6). In contrast to most other gram- pm. They also appear to be S shaped and gull winged when negative bacteria, campylobacters do not contain two cells form short chains. Nonsporeforming. Cells in old ubiquinones as major isoprenoid quinones but do contain cultures may form spherical or coccoid bodies. Motile (with menaquinones. All of the Campylobacter strains belonging a characteristic corkscrewlike, darting motion) by means of to rRNA cluster I (including Campylobacter curvus and a single polar unsheathed flagellum at one or both ends of the Campylobucter rectus) that were tested have menaquinone 6 cell. Microaerophilic to anaerobic with a respiratory type of and methyl-substituted menaquinone 6 as their major iso- metabolism. Growth at 37°C; no growth at 15°C. An 0, prenoid quinones (35, 36). Studies of the cellular fatty acid concentration of 3 to 15% is required for the preferentially compositions of these organisms revealed that they all have microaerophilic species, while most strains (Campylobacter the same major fatty acids (i.e., tetradecanoic acid, hexade- curvus and Campylobacter rectus) which prefer to grow canoic acid, hexadecenoic acid, and octadecenoic acid [21, under anaerobic conditions can also grow in the presence of 281) (data for Campylobacter curvus and Campylobacter 1 to 5% 0,. Hydrogen is required or stimulates growth under rectus were obtained from the database of the Microbial both microaerophilic and anaerobic conditions. Occasionally Identification System described by Miller [33]). Most cam- a few strains may grow slightly under aerobic conditions pylobacters have DNA base ratios in the range from 30 to 41 (20% 0,). Chemoorganotrophs. Carbohydrates are neither mol% G+C, while Campylobacter curvus, Campylobacter oxidized nor fermented. Does not require serum or blood for rectus, and [Wolinella] sp. strain CCUG 11641 have G+C growth. Energy is obtained from amino acids or tricarboxy- contents between 45 and 46 mol% (Table 2). This G+C lic acid cycle intermediates and not from carbohydrates. content range is rather large but not unique in bacterial Urea (except for a group of atypical Campylobacter lari taxonomy (39). On the basis of the numerous genotypic and strains [4]) and gelatin are not hydrolyzed. Oxidase activity, phenotypic similarities, we conclude that the genus Cam- but no lipase activity. No pigment production, except for pylobacter should be restricted to the Campylobacter spe- Campylobacter mucosalis and Campylobacter hyointestina- cies belonging to rRNA cluster I and that the former species lis, which produce a dirty yellow pigment. Menaquinone 6 [ Wolinella] curva and [ Wolinella] recta and [ Wolinella] sp. and methyl-substituted menaquinone 6 are the major respi- strain CCUG 11641 should be included in the genus Cam- ratory quinones ; tetradecanoic acid, hexadecanoic acid, pylobacter as Campylobacter curvus, Campylobacter rec- hexadecenoic acid, and octadecenoic acid are the major fatty tus, and Campylobacter sp. incertae sedis, respectively. acids. The G+C content of the DNA ranges from 30 to 46 Tanner and co-workers found no significant levels of DNA mol%. homology between Campylobacter curvus and Campylobac- Several species are pathogenic for humans and animals. ter rectus and between Campylobacter curvus and Campylo- Found in the reproductive organs, intestinal tracts, and oral bacter sp. strain CCUG 11641; DNA homology values cavities of humans and animals. The type species is Cam- ranging from 34 to 55% were found between Campylobacter pylobacter fetus (Smith and Taylor 1919) Sebald and Veron rectus and Campylobacter sp. strain CCUG 11641 (54, 55, 1963. 57). Campylobacter curvus and Campylobacter rectus are This emended genus contains the following species: Cam- immunologically more closely related to each other than to pylobacter fetus, Campylobacter hyointestinalis, Campylo- the other Campylobacter taxa (Table 4). bacter mucosalis, Campylobacter concisus, Campylobacter Emendation of the genus Campylobacter. In Bergey ’s Man- sputorum, Campylobacterjejuni, Campylobacter coli, Cam- ual of Systematic Bacteriology, the genus Campylobacter pylobacter lari, “Campylobacter upsaliensis,” Campylo- consists of the following five species: Campylobacterfetus, bacter curvus, and Campylobacter rectus. All other Cam- Campylobacter jejuni, Campylobacter coli, Campylobacter pylobacter species are generically misnamed. sputorum, and Campylobacter concisus (48). DNA-DNA Description of Campylobacter curvus comb. nov. Campylo- hybridization studies showed that Campylobacter sputorum bacter curvus (basonym, Wolinella curva Tanner, Listgar- subsp. mucosalis represents a separate species (i.e., Cam- ten, and Ebersole 1984) (curv’us. L. adj. curvus, curved). pylobacter mucosalis) and that Campylobacter sputorum The description is the same as that given previously for subsp. sputorum, Campylobacter sputorum subsp. bubulus, [ Wolinella] curva (55). and “Campylobacter fecalis” (17) should be considered Description of Campylobacter rectus comb. nov. CampyLo- biovars of Campylobacter sputorum (43, 44). Our DNA- bacter rectus (basonym, Wolinella recta Tanner, Badger, rRNA hybridization results confirm that Campylobacter lari, Lai, Listgarten, Visconti, and Socransky 1981) (rect’us. L. Campylobacter hyointestinalis, and “Campylobacter upsa- adj. rectus, straight). The description is the same as that liensis” are true Campylobacter species (29, 39, 42, 58). given previously for [ Wolinella] recta (54, 56). Recently, two groups of CLO strains, the nitrate-negative Taxonomic position of generically misnamed Bacteroides Campylobacter strains (25, 50) and the urease-positive ther- species. The taxonomic position of [Bacteroides]gracilis and mophilic Campylobacter strains (3, 4), were shown to be [Bacteroides] ureolyticus remains uncertain. These organ- atypical Campylobacter jejuni and Campylobacter lari isms share a number of phenotypic characteristics with and strains, respectively (38, 51). The name Campylobacter have DNA base ratios in the range proposed for the emended jejuni subsp. doylei was given to the nitrate-negative Cam- genus Campylobacter (Table 2) (24, 39, 54). The fatty acid pylobacter strains (51). Several of these recently described composition of [Bacteroides] gracilis resembles the fatty VOL.41, 1991 TAXONOMY OF rRNA SUPERFAMILY VI 99

TABLE 6. Characteristics that differentiate between the genus Arcobacter and related genera belonging to rRNA superfamily VI

Presence Growth at: of hexa- Nitrate Growth Hydrol- Cell morphol- No. of Position of Flagellar G+C Major decanoic Genus reduc- on 0.5% ysis of flagella flagella sheaths content isoprenoid acid as a tion glycine urea OgY (mol%) quinones major 15°C 30°C 42°C compo- nent

Arcobacter +‘ ND V + + - Curvedand 1 Polar Absent 28-31 MK-6, Un-MK-6 +b spiral rods Campylobacter‘ +d V - e- + V Curved and 1 Polar Absent 30-46 MK-6, *MK-6 + spiral rods Wolinelld + - - - - W Spiral 1 Polar Absent 47 MK-6, *MK-6 + Helicobactefl V + V - V V Curved and V Polar and Present 35-44 MK-6, Un-MK-6 - spiral rods lateral “Flexispira” - + + -- + Fusiform Multiple Polar Present 33 ND ND rods

‘‘ Data from references 1, 19-21, 28, 32, 35-37, 48, and 56 and from our study. f, Reaction is positive for 90% of the strains; -, reaction is negative for 90% of the strains; V, variable reactions for different species; W. weak reaction; MK-6, menaquinone 6; *MK-6, methyl-substituted menaquinone 6; Un-Mk-6, substituted menaquinone with unknown structure; ND, not determined. Data for A. cryaerophilus only. ‘ Emended genus Campylobacter. All members of the genus except C.jejuni subsp. doylei. All members of the genus except the urease-positive thermophilic variants of C. luri (3, 4). The genus Wolinellu contains only one species, W. succinogenes. Emended genus Helicobacter.

acid compositions of the authentic campylobacters; [Bac- members of rRNA cluster I1 (Table 2) and by their pheno- teroides] ureolyticus has a considerably different fatty acid typic (Table 5) and immunological (Table 4) characteristics. composition, with hexadecenoic acid, 3-hydroxydecanoic No significant DNA binding values were measured between acid, octadecanoic acid, and octadecenoic acid as the major Arcobacter nitrojigilis and Arcobacter cryaerophilus (Fig. fatty acids (data are from the database of the Microbial 2). The fact that two species of one genus have such diverse Identification System, as described by Miller [33]). Our habitats (Arcobacter cryaerophilus occurs in animal and DNA-rRNA hybridization results confirm that these organ- human hosts [37], whereas Arcobacter nitrojigilis is a nitro- isms are closely related to the campylobacters (39), but gen-fixing, plant-associated bacterium [32]) is not unique in additional data are required before definite conclusions can bacterial taxonomy. Similar situations occur in several other be drawn. genotypically coherent genera (e.g., in the genera Xanth- rRNA cluster 11. rRNA cluster I1 is a rather homogeneous ornanas [52] and Cornarnonas [53]). Strain CCUG 10373 has and clearly separated rRNA cluster (Fig. 1). We propose that a separate position on the Tm(e)dendrogram (Fig. 1) and the organisms in this cluster should be included in a new exhibited no significant DNA binding with the other organ- genus, Arcobacter, with two species, Arcobacter nitrojigilis isms belonging to the genus Arcobacter (Fig. 2). Strain and Arcobacter cryaerophilus. These two species and CLO CCUG 10373 is immunologically (Table 4) and phenotypi- strains CCUG 10373, CCUG 10374, and CCUG 10375 have cally (Table 5) different from Arcobacter nitrojigilis, Arco- similar G+C values (28 to 30 mol%) (Table 2) and share a bacter cryuerophilus and CLO strains CCUG 10374 and considerable number of phenotypic characteristics (see be- CCUG 10375. Additional strains should be isolated and low). The genus Arcobacter can be differentiated on the studied before an appropriate species description can be basis of phenotypic characteristics from the other genera of proposed. We believe that CLO strain CCUG 10373 is an rRNA superfamily VI (Table 6). Arcobucter nitrofigilis and Arcobacter strain. CLO strains CCUG 10374 and CCUG Arcobacter cryaerophilus can be differentiated from each 10375 are closely related to each other (96% DNA binding) other by their Tm(e, values versus reference rRNAs from (Fig. 2). Although their average ?‘m(e) values versus two reference rRNAs from members of rRNA cluster I1 were approximately 1.5”C lower than those of Arcobacter CCUG cryaerophilus strains (Table 2 and Fig. l), these two strains No. seem to be more closely related to Arcobacter cryaerophilus than to Arcobacter nitrofigilis or CLO strain CCUG 10373. They had an average DNA binding value versus DNAs from Arcobacter cyaerophilus strains of 36% (Fig. 2) and are phenotypically (Tables 4 and 6) similar to this species. For the time being, we believe that these two strains belong to the genus Arcobacter. The description of Arcobacter gen. nov. below is based on data from Neil1 et al. (37), McClung FIG. 2. DNA-DNA hybridization results for nine Arcobacter et al. (32), Moss et al. (36), and Han et a]. (23). strains. Each DNA-DNA hybridization value is the average degree Description of Arcobacter gen. nov. Arcobacter (Ar’co. of binding from at least two experiments. Three DNA homology bac.ter. L. n. arcus, bow; Gr. n. bacter, rod; M. L. masc. n. groups (with DNA bindig values of more than 46%) and one separate Arcobacter, bow-shaped rod) cells are grarn-negative non- strain are clearly delineated. sporeforming rods (width, 0.2 to 0.9 pm; length, 1 to 3 Fm) 100 VANDAMME ET AL. INT. J. SYST.BACTERIOL. that are usually curved, S shaped, or helical. Motile (with a pylobacter] cinaedi and [Campylobacter] fennelliae have darting, corkscrewlike motion) by means of a single polar, tetradecanoic acid, hexadecanoic acid, and octadecenoic unsheathed flagellum. Growth occurs at 15,30, and 37°C; no acid as major fatty acids, while Helicobacter pylori and growth occurs at 42°C. Optimal growth occurs under mi- Helicobacter mustelae have an additional 19-carbon cyclo- croaerophilic conditions (3 to 10% 02).Hydrogen is not propane fatty acid as a major fatty acid component) also required for microaerophilic growth. Growth occurs in the differentiate [Campylobacter] cinaedi and [Campylobacter] presence of 1 and 2% NaCl and 1% (wt/vol) pteridine fennelliae from Helicobacter pylori and Helicobacter mus- vibriostatic compound 0/129. No growth occurs in the pres- telae. However, we believe that the genotypic and pheno- ence of 1% glycine and 0.1% 2,3,5-triphenyltetrazolium typic similarities of the two Helicobacter species, [Campylo- chloride. The strains have oxidase and catalase activities and bacter] cinaedi, and [Campylobacter]fennelliae outweigh reduce nitrate. Negative reactions in methyl red and Voges- their differences and justify inclusion of [Campylobacter] Proskauer tests. Indole is not produced. Carbohydrates are cinaedi and [Campylobacter]fennelliae in an emended genus neither fermented nor oxidized. Organic and amino acids are Helicobacter. utilized as carbon sources. Hippurate, esculin, starch, and Emended description of the genus Helicobacter Goodwin, DNA are not hydrolyzed; gelatin is not liquified. Nonhemo- Armstrong, Chilvers, Peters, Collins, Sly, McConnell, and lytic. Menaquinone 6 and a second atypical menaquinone 6, Harper 1989. Helical, curved, or straight unbranched gram- the identity of which remains to be established, are the major negative cells that are 0.3 to 1.0 pm wide and 1.5 to 5 pm respiratory quinones. long and have rounded ends and spiral periodicity. Non- Strains have been isolated from root-associated sediments sporeforming. Cells in old cultures may form spherical or and roots of salt march plants, from aborted fetuses of coccoid bodies. Darting motility by means of a single polar several species of farm animals, and from various other flagellum (Helicobacter cinaedi and Helicobacter fennelliae) animal and human sources. Pathogenicity is unknown. or multiple unipolar or bipolar and lateral flagella (Helico- The type species is Arcobacter nitrofigilis comb. nov. The bacter pylori and Helicobacter mustelae). Flagella are DNA base composition ranges from 28 to 31 mol% G+C. sheathed (20, 23). Microaerophilic with a respiratory type of Description of Arcobacter nitrofigilis comb. nov. Arcobacter metabolism. Chemoorganotrophs. Carbohydrates are nei- nitrofigilis (basonym, Campylobacter nitrofigilis McClung, ther oxidized nor fermented. Energy is obtained from amino Patriquin, and Davis 1983) (ni. tro. fig’i. lis. L. n. nitrum, acids or tricarboxylic acid cycle intermediates but not from nitrate; L. v. figo, to fix; L. adj. suff.-ilis,able to; M. L. adj. carbohydrates. Optimal growth occurs at 37°C in a humid nitrofigilis, able to fix [nitrogen] as nitrate). The description atmosphere; no growth occurs at 25°C. Hydrogen is required is the same as that given previously for Campylobacter or stimulates growth. No growth occurs in the presence of nitrofigilis (32). 3.5% NaCl. Growth occurs in the presence of 0.5% glycine Description of Arcobacter cryaerophilus comb. nov. Arco- and 0.04% triphenyltetrazolium chloride. Catalase and oxi- bacter cryaerophilus (basonym, Campylobacter cryaero- dase activities are present. No pigment production. No H,S phila Neill, Campbell, O’Brien, Weatherup, and Ellis 1985) production in triple sugar iron agar. No hydrolysis of hippu- (cry. ae. ro’ phi.lus. Gr. n. cruos, cold; Gr. n. aer, air; Gr. n. rate. Susceptible to ampicillin, gentamicin, rifampin, and philos, friend; M. L. adj. cryaerophilus, friend of cold and tetracycline; resistant to trimethoprim. Variable resistance air). The description is the same as that given previously for to nalidixic acid and cephalothin (18, 20). The G+C content Campylobacter cryaerophila (37). of the DNA ranges from 35 to 44 mol%. rRNA cluster 111. rRNA cluster I11 contains members of Isolated from gastric mucosa of humans and animals, from four different genera (Helicobacter, Wolinella, “Flexispira,” blood and feces of homosexual males, and from intestines of and misnamed Campylobacter species) and strain CLO-3 of hamsters. Some organisms in this genus may be associated Fennell et al. (16). Wolinella succinogenes has a separate with gastritis and peptic ulcers in humans. position on the TmCe)dendrogram (Fig. 1). The other taxa The type species is Helicobacter pylori. belonging to this rRNA branch have AT,,,, values of 6 to Description of new Helicobacter species. The descriptions of 10°C versus Wolinella succinogenes rRNA (Table 2). Fur- Helicobacter cinaedi comb. nov. (basonym, Campylobacter thermore, isoprenoid quinone composition (36) and several cinaedi Totten, Fennell, Tenover, Wezenberg, Perine, other characteristics (20) separate Wolinella succinogenes Stamm, and Holmes 1985) and Helicobacter fennelliae from the other taxa of this rRNA homology group. Strain comb. nov. (basonym, Campylobacter fennelliae Totten, CLO-3 is the only organism with a DNA base ratio similar to Fennell, Tenover, Wezenberg, Perine, Stamm, and Holmes that of Wolinella succinogenes (Table 2); however, this is 1985) are the descriptions given by Totten et al. (59) for not necessarily an indication of a close relationship (9). [Campylobacter] cinaedi and [Campylobacter] fennelliae, Goodwin et al. (20) transferred [Campylobacter] pylori respectively. and [Campylobacter]mustelae to the new genus Helicobac- Taxonomic position of ‘‘Flexispira rappini” and strain ter. The DNA-rRNA hydbridization results (Table 2 and Fig. CLO-3. “Flexispira rappini” and strain CLO-3 also belong to 1) indicate that Helicobacter pylori, Helicobacter mustelae, rRNA cluster I11 (Fig. 1). No significant precipitation values [Campylobacter] cinaedi, and [Campylobacter]fennelliae were found in the immunotyping analysis versus antisera of are related to each other, at least at a level similar to the level other taxa belonging to rRNA cluster I11 (Table 4). “Flex- between Campylobacter fetus and Campylobacter jejuni in ispira rappini” has sheathed flagella and a DNA base com- rRNA cluster I (Fig. 1). Furthermore, all four species have position similar to the base compositions of Helicobacter G+C values between 35 and 41 mol% (Table 2) (20) and have strains (33 to 35 mol%) (Table 2). However, its unusual many phenotypic characteristics in common (16, 20, 23, 59). ultrastructure (spiral grooves) and several other phenotypic Immunotyping (Table 4) and partial 16s rRNA sequence traits clearly distinguish this organism from the other taxa analysis (58) revealed that [Campylobacter] cinaedi and belonging to rRNA cluster 111 (20). Strain CLO-3 has a DNA [Campylobacter]fennelliae are more closely related to each base ratio similar to that of Wolinella succinogenes (Table other than to Helicobacter pylori or Helicobacter mustelae. 2), but a ATm(e)of 9°C versus the latter organism (Fig. 1 and The number of flagella and fatty acid composition ([Cam- Table 2). Strain CLO-3 is also phenotypically different from VOL. 41, 1991 TAXONOMY OF rRNA SUPERFAMILY VI 101 the other organisms of rRNA cluster I11 (20, 59). The measurement of DNA hybridization from renaturation rates. relationships of strain CLO-3 and “Flexispira rappini” to Eur. J. Biochem. 12:133-142. the other taxa of rRNA cluster I11 remain to be investigated. 11. De Ley, J., and J. De Smedt. 1975. Improvements on the Free-living Campylobacter-like strains. The saprophytic membrane filter method for DNA:rRNA hybridization. Antonie van Leeuwenhoek J. Microbiol. Serol. 41:287-307. campylobacters of Laanbroek et al. (27) and Wolfe and 12. De Ley, J., P. Segers, and M. Gillis. 1978. Intra- and intergeneric Pfennig (65) do not belong to one of the three major rRNA similarities of Chrornobacteriurn and Janthinobacteriurn ribo- homology groups and thus cannot be included within one of somal ribonucleic acid cistrons. Int. J. Syst. Bacteriol. 28:154- the five genera described above (Table 2 and Fig. 1). The 168. DNA base ratios of [Spirillurn] sp. strain 5175 and Laan- 13. De Vos, P., and J. De Ley. 1983. Intra- and intergeneric broek strain CCUG 13942 are 38.4 and 41.6 mol%, respec- similarities of Pseudomonas and Xanthornonas ribosomal ribo- tively (27, 65). Both organisms are sulfur-reducing bacteria nucleic acid cistrons. Int. J. Syst. Bacteriol. 33:487-509. with a similar type of metabolism and are believed to belong 14. De Vos, P., K. Kersters, E. Falsen, B. Pot, M. Gillis, P. Segers, to a group of saprophytic Campylobacter-like organisms and J. De Ley. 1985. Cornarnonas Davis and Park 1962 gen. which are isolated frequently from sediment samples of dirty nov., nom. rev. emend., and Cornarnonas terrigena Hugh 1962 sp. nov., nom. rev. Int. J. Syst. Bacteriol. 35443453. freshwater or brackish water (41). Like all other members of 15. Falsen, F. 1983. Immunodiffusion as an aid in routine identifi- rRNA superfamily VI, [Spirillurn] sp. strain 5175 has mena- cation of uncommon aerobic Gram negative bacteria, p. 477- quinone 6 as one of its major respiratory quinones (7). As 483. In H. Leclerc (ed.), Gram negative bacteria of medical and strain 5175 and strain CCUG 13942 are genotypically and public health importance: taxonomy, identification, applica- phenotypically similar (41) and have similar taxonomic po- tions. Les editions de I’Institut National de la SantC et de la sitions within rRNA superfamily VI (Fig. l),it does not seem Recherche Medicale, Paris. unlikely that they belong to a single, separate genus within 15a.Falsen, E., L. Nehls, and A. Borjesson. 1986. Abstr. XIV Int. rRNA superfamily VI. Cong. Microbiol., Manchester, United Kingdom, 0.B8-4, p. 8. 15b.Falsen, E., L. Nehls, and A. Borjesson. 1986. Abstr. XIV Int. Cong. Microbiol., Manchester, United Kingdom, P.B8-8, p. 60. 16. Fennell, C. L., P. A. Totten, T. C. Quinn, D. L. Patton, K. K. ACKNOWLEDGMENTS Holmes, and W. E. Stamm. 1984. Characterization of Carnpylo- bacter-like organisms isolated from homosexual men. J. Infect. 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