INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, Jan. 1991, p. 88-103 0020-7713/91/010088-16$02.00/0 Copyright 0 1991, International Union of Microbiological Societies

Vol. 41, No. 1

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 hyointestinalis, 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.

Dewhirst (39) found a close relationship between Wolinella curva, Wolinella recta, Bacteroides gracilis, and Bacteroides ureolyticus on the one hand and the so-called true campylobacters (i-e., the Campylobacter taxa belonging to rRNA homology group I of Thompson et al. [58] on the other. These Wolinella and Bacteroides species have been isolated from humans with both oral and nonoral infections (39). It was the aim of this study to include all known campylobacters and possible relatives in a single phylogenetic study. Therefore, we selected reference strains of the 15 Campylobacter and Helicobacter species, representative strains of the saprophytic campylobacters (i.e. , the aspartate-fermenting, free-living Campylobacter species of Laanbroek et al. [27] and Spirillum sp. strain 5175 of Wolfe and Pfennig [65]), and strain CLO-3 of Fennel1 et al. (16). In addition to these Campylobacter strains, we also included representative strains of Wolinella succinogenes, Wolinella curva, and Wolinella recta, and an unnamed Wolinella strain (57), as well as representative strains of Bacteroides gracilis, Bacteroides ureolyticus, and “Flexispira rappini,” which is a 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 organisms and their phylogenetic relationships with other gramnegative bacteria, we used the DNA-rRNA hybridization

At present, the genus Campylobacter consists of 13 welldefined species (40). Recently, two additional species, Campylobacter pylori and Campylobacter mustelae, were included in the new genus Helicobacter as Helicobacter pylori and Helicobacter mustelae, respectively (20). The clinical significance of all of these organisms was reviewed recently by Penner (40).A study of the taxonomic structure of the genus Campylobacter in which partial 16s rRNA sequence analysis was used revealed that the Campylobacter species can be divided into three major rRNA homology groups (58). The first homology group contains Campylobacterfetus (the type species of the genus Campylobacter), Campylobacter hyoin testina h , Campyloba cter sp u torum , Campyloba cter jejuni, Campylobacter coli, Campylobacter lari (62), “Campylobacter upsaliensis,” Campylobacter concisus, and Campylobacter mucosalis. The second rRNA homology group contains Campylobacter pylori, Campylobacter fennelliae, and Campylobacter cinaedi; Wolinella succinogenes, an organism found in bovine rumina, also belongs to this second rRNA homology group. The third rRNA homology group consists of Campylobacter nitrojigilis and Campylobacter cryaerophila. Furthermore, in another phylogenetic study of the genus Campylobacter, Paster and

* Corresponding author. t Present address: Innogenetics N.

V., Antwerp, Belgium. 88

VOL.41, 1991

TAXONOMY OF rRNA SUPERFAMILY VI

technique of De Ley and De Smedt (11) and the immunotyping technique of Falsen (15). This enabled us to study several strains of most taxa, whereas only single representative strains have been included in previous partial 16s rRNA sequence analyses (29, 39, 42, 58). Below, we use the new names Campylobacter curvus, Carnpylobacter rectus, Arcobacter nitrojigilis, Arcobacter cryaerophilus, Helicobacter cinaedi, and Helicobacter fennelliae for the organisms formerly called Wolinella curva, Wolinella recta, Carnpylobacter nitrojigilis, Carnpylobacter cryaerophila, Carnpylobacter cinuedi, and Campylobacter fennelliae, respectively. (Preliminary immunotyping results have been presented previously [15a, 15bl.) MATERIALS AND METHODS Bacterial strains and growth conditions. All of the strains used for DNA-DNA hybridization and DNA-rRNA hybridization experiments and the representative strains used for the immunotyping analysis are shown in Table 1. Names in quotation marks have not been validly published. Below, the genus names of all generically misnamed taxa are enclosed in brackets. Bacteriological purity was checked by plating and examining living and Gram-stained cells. For mass cultures, cells were grown on petri dishes on blood agar media containing 5% (voVvol) horse blood and solidified with 1.8% agar. All Wolinella, Bacteroides, and saprophytic Campylobacter strains were incubated at 37°C in an anaerobic atmosphere containing 5% CO,, 10% H2, and 85% N,. All other strains except the Arcobacter nitrofigilis strains were grown in a microaerophilic atmosphere containing approximately 5% 0,, 3.5% CO,, 7.5% H,, and 84% N, at 37°C; the cultures of Arcobacter nitrofigilis strains were incubated at 30°C. The strains to be used as antigens were incubated in a microaerophilic atmosphere containing approximately 4% C 0 2 , 7% O,, 27% N,, and 62% H,. Preparation of high-molecular-weight DNAs. High-molecular-weight native DNAs were prepared from 0.5 to 2 g (wet weight) of cells by using the method of Marmur (30), purified by CsCl gradient centrifugation, and stored at -80°C. Preparation of radioactively labeled rRNAs. Shake cultures of Campylobacter fetus subsp. fetus CCUG 6823AT (T = type strain), Campylobacter sputorum biovar bubulus CCUG 11289, and Campylobacter coli CCUG 11283T were grown in nutrient broth no. 2 (catalog no. CM67; Oxoid Ltd., Basingstoke, United Kingdom). Shake cultures of Carnpylobacter concisus CCUG 20534 and Arcobacter nitrofigilis CCUG 12022 were grown in the same broth medium supplemented with 0.2% (wthol) sodium formate and 0.3% (wthol) disodium fumarate (for Campylobacter concisus CCUG 20534) or 1% (wthol) NaCl (for Arcobacter nitrofigilis CCUG 12022). Shake cultures of Wolinella succinogenes CCUG 13145T and Campylobacter-like organism (CLO) strain CCUG 10373 were grown in the medium of Wolin et al. (66). All cultures were incubated for 1 to 3 days under the appropriate atmospheric and temperature conditions (see above); 2 mCi of 2,8-C3H]adenine was added to 100 ml of a culture in the early log phase of growth for CLO strain CCUG 10373, and 1 to 2 mCi of 5,6-[3H]~racilwas added for the other strains. Labeled rRNAs were prepared and separated into 23s and 16s fractions as described by De Ley and De Smedt (11). The specific activities of the 23s rRNA fractions were as follows: 14,000 cpm/pg for Campylobacter fetus subsp. fetus CCUG 6823AT; 115,000 c p d p g for Campylobacter sputorum biovar bubulus CCUG 11289; 65,000

89

cpmtpg for Campylobacter concisus CCUG 20534; 2,000 cpm/pg for Arcobacter nitrofigilis CCUG 12022; 70,000 cpm/pg for CLO strain CCUG 10373; and 29,000 cpm/pg for Wolinella succinogenes CCUG 13145T. These specific activities were determined with a Beckman model 3310 Tri-Carb scintillation counter. DNA-rRNA hybridization experiments. Fixation of singlestranded DNAs on membrane filters, chemical determination of the amounts of DNAs on the filters, saturation hybridization, RNase treatment, and measurement of the thermostabilities of the hybrids were performed as described previously (11).We used labeled 23s rRNAs from Cumpylobacter, Arcobacter, and Wolinella strains (Table 2) and a variety of gram-negative reference strains (Table 3). Each DNA-rRNA hybrid was characterized by the following two parameters: (i) the melting temperature of elution [T,(,J, which was the temperature at which 50% of a DNA-rRNA hybrid was denatured; and (ii) the percentage of rRNA binding, which was a way to measure the amount of labeled rRNA bound to 100 pg of filter-fixed DNA after RNase treatment under standard conditions. A homologous duplex was formed between the DNA and the rRNA of the same strain; a heterologous hybrid was formed between DNA and rRNA of different strains. The T,(,, is the most important parameter for drawing taxonomic conclusions (11, 13); the higher the T,(,, of a heterologous hybrid, the more closely the two strains are related. The Tm(e) values were used to calculate the average linkage level between each pair of rRNA branches by using the unweighted average pair group method (49). The percentage of rRNA binding can be useful for distinguishing strains with similar T,(,) values. The difference between the T,(,, of a homologous duplex and the Tmc,)of a heterologous hybrid is called the AT,,,,. DNA-DNA hybridization experiments. The degree of DNADNA binding, expressed as a percentage, was determined spectrophotometrically by using the initial renaturation rate method of De Ley and co-workers (10). Each value given below is the average of the values from at least two hybridization experiments. DNA binding values of 30% or less indicate that there is no significant DNA homology. The total DNA concentration was about 39 pg/ml, and the optimal SSC is 0.15 M NaCl renaturation temperature in 2~ SSC (Ix plus 0.015 M sodium citrate, pH 7) was 64.6"C. DNA base compositions. All of the guanine-plus-cytosine (G+ C) values were determined by the thermal denaturation method and were calculated by using the equation of Marmur and Doty (31), as modified by De Ley (8). Immunotyping. Preparation of soluble, cell wall-free antigens and immunization and immunodiffusion experiments were performed as described previously (15, 61). Phenotypic tests. Phenotypic tests were performed with CLO strains CCUG 10373, CCUG 10374, and CCUG 10375 as described previously (22). RESULTS DNA base compositions. Within the species Campylobacter concisus we found a G + C range of 37.9 to 40.4 mol% (Table 2), which is similar to the results of Roop et al. (43) but higher than the range of values (34 to 38 mol%) found by Tanner et al. (54). All of the other DNA base ratios which we determined (Tables 2 and 3) agree with previously published data (2, 24, 48, 55, 56). DNA-rRNA hybridization experiments. Table 3 shows the results of the DNA-rRN A hybridizations between DNAs from Campylobacter and Arcobacter strains and radioac-

TABLE 1. Strains used Strain”

Organisms belonging to rRNA cluster I Campylobacter fetus subsp. fetus CCUG 6823AT C. fetus subsp. fetus ATCC 33246 C. fetus subsp. fetus NIDO 7572 C. fetus subsp. fetus NIDO 212514 C. fetus subsp. venerealis CCUG 538T C. fetus subsp. venerealis NIDO 483 C. fetus B5 C . hyointestinalis CCUG 14169T C. hyointestinalis CCUG 14916 C. hyointestinalis ADRI 1047 C. hyointestinalis CCUG 20823 C . concisus CCUG 13144T C. concisus CCUG 20534 C. concisus CCUG 17580 C. concisus CCUG 18688 C. concisus CCUG 19219 C. miicosalis CCUG 6822T C . mucosalis CCUG 10771 C. mucosalis FS 921177 CLO strain CCUG 20705 Campylobacter sputorum biovar sputorum CCUG 9728T C. sputorum biovar bubulus CCUG 11289 C. sputorum biovar bubulus CCUG 886 C. sputorum biovar bubulus ATCC 33491 C. sputorum biovar fecalis CCUG 12015 C. sputorum biovar fecalis CCUG 12017 C. sputorum biovar fecalis CCUG 17761 C. sputorum biovar fecalis RB6t2 C. cofi CCUG 11283T C. coli CCUG 8169 C. coli CCUG 10369 C. jejuni subsp. jejuni ATCC 33250 C. jejuni subsp. jejuni CCUG 14914 C. jejuni subsp. jejuni CCUG 6824 C . jejuni subsp. jejuni RV4 C. jejuni subsp. jejuni CCUG 10370 C. jejuni subsp. jejuni M2 C. jejuni subsp. jejuni JJ91 C. jejuni subsp. doylei CCUG 18265 C. lari NCTC 11352T C. lari CCUG 12774 C . lari CCUG 18267 “C. upsaliensis” CCUG 14913 “C. upsaliensis” B523 “C. upsaliensis” E282 “C. upsaliensis” CCUG 20818 C. curvus CCUG 13146T C. rectus CCUG 20446T C . rectus CCUG 11640 [Wolinella]sp. strain CCUG 11641 [Bacteroides]gracilis FDC 404 [Bacteroides]ureolyticus CCUG 7319T [Bacteroides]ureolyticus CCUG 9596 [Bacteroides]ureolyticus CCUG 9510D Organisms belonging to rRNA cluster I1 Arcobacter nitrofgilis CCUG 15893T A . nitrofgilis CCUG 12022 A . cryaerophilus CCUG 17801T A . cryaerophilus CCUG 17805 A . cryaerophilus CCUG 12018 A . cryaerophilus CCUG 12019 CLO strain CCUG 10373 CLO strain CCUG 10374 CLO strain CCUG 10375

Other designations“

Received from“:

Source

NCTC

Brain of sheep fetus Joint aspirate Calf fetus Genitals of a bull Vaginal mucus of a heifer Bovine

LMG 7817T, Gebhart 80-4577-4T LMG 7538, C 269 LMG 8634, CCUG 24181 LMG 8216, CDC D2189 LMG 77!BT, FDC 484T LMG 7789, NCTC 11486 LMG 7545 LMG 7963

Mannheim Dekeyser Deke y ser NCTC Deke y ser Dekey ser Gebhart Ursing Garcia Patton Tanner NCTC Tornqvist Jansson

LMG LMG LMG LMG

Goodwin NCTC NCTC Lawson

LMG 6442T, N%TC 10842T LMG LMG LMG LMG

6569, CCUG 17693 6571, CCUG 17694 6443T, NCTC 10354T 6570, CCUG 7477

7964, Goodwin 13961 6448T, NCTC llOOOT 7794, NCTC 11001 8499, CCUG 23201

LMG 7974, Bolton A4 LMG 7795T, VPI S-17T

Bolton Holdeman

LMG 6447, CIP 53103 NCTC 10355

CIP NCTC Mannheim Karmali Karmali NCTC Dekey ser CIP

LMG LMG LMG LMG LMG

6617, PC 363 6618, PC 365 8531, NCTC 11415 6728, CCUG 17695B 6440T, CIP 7080T

LMG 7535, Skirrow 4620178 LMG LMG LMG LMG

7534 8553, NCTC 11168 6629, CCUG 17696 6446

LMG 7790, NCTC 11847 LMG 8846T, CCUG 23947T LMG 7607, Skirrow 175182 LMG 7791, NCTC 11845 LMG 8850, NCTC 11541 LMG 8852, CCUG 24191 LMG 7917 LMG 7915, CDC D533 LMG 7609T, VPI 9584T NCTC 11489T,FDC 371T LMG 7611, D13a-g, VPI 10278B LMG 8543, VPI 10279 LMG 7616, CCUG 22762 LMG 6451T, NCTC 10941T

Skirrow Mannheim Ursing NCTC Dekeyser Skirrow Mannheim Mannheim NCTC NCTC Skirrow NCTC Ursing Goossens Goossens Patton Tanner NCTC Sundqvist Sundqvist Tanner NCTC Our own isolate Our own isolate

LMG 7604T, CCUG 15892T, CI’ LMG 7547, PC 371

McClung Karmali

LMG LMG LMG LMG LMG LMG LMG

Neill Neill Karmali Karmali Skirrow Skirrow Skirrow

7536T, Neill A1691BT 7537, Neill B1056lP 6622, PC 367 9065, Neill 02797 6620, Skirrow 996/79 6621, Skirrow 449180 8538, Skirrow 1018179

Porcine intestine Bovine feces Feces of a diarrheic beef calf Human stool Gingival sulcus Periodontal pocket Diarrheic feces of 2-yr-old child Feces from woman suffering from fever and diarrhea Esophagus biopsy specimen Porcine small intestine Porcine intestine Colon of pig with proliferative enteropathy Porcine intestine Human mouth Bull sperm Semen of normal bull Ovine feces Ovine feces Ovine feces Ovine feces Porcine feces Human Porcine placenta Human blood Canine feces Human feces Human

Human gastric biopsy Cloaca1 swab of a herring gull Child, feces River water Canine feces Human feces Human feces Human feces Human alveolar abscess Human periodontitis Human dental root canal Human dental root canal Gingival crevice Amniotic fluid Wound, penis Wound, penis Roots of Spartina alternifora Roots or root-associated sediment of Spartina alternijlora Aborted bovine fetus Aborted ovine fetus Kidney of an aborted porcine fetus Placenta of aborted ovine fetus Human blood Feces of lamb with diarrhea Bovine Continued on following page

90

TAXONOMY OF rRNA SUPERFAMILY VH

VOL. 41, 1991

91

TABLE 1-Continued Strain“

Organisms belonging to rRNA cluster 111 Helicobacter cinaedi CCUG 18818T H . cinaedi CCUG 15432 H . cinaedi CCUG 17733 H . fennelliae CCUG 18820T H . pylori CCUG 17874T H . pylori CCUG 15816 H . pylori CCUG 19106 H . mustelae CCUG 23652 “Flexispira rappini” ATCC 43879

“ F . ruppini” ATCC 43880 Wolinella succinogenes CCUG 13145T W . succinogrnes DSM 1740T CLO-3 strain CCUG 14564 Other campylobacters and reference strains CLO strain CCUG 13942 [Spirillurn] sp. strain 5175 Actinobacillus lignieresii NCTC 4189T Agrobacterium tumefuciens ICPB T T l l l Aquaspirillum aqmticxm ATCC 11330T Aquaspirillum serpens ATCC 12638T Arthrobucter oxydans CBRI 21010T Bacteroides coagulans CCUG 10974T Bacteroides f r a g i h NCTC 9345 Bacteroides orulis ATCC 33269= Bacteroides splanchnicus ATCC 29572= Brucella ubortus ATCC 23448T Brucella melitensis NCTC 10094T Cardiobucterium hominis ATCC 15826T Cytophaga johnsnnue ATCC 1706IT Escherichia coli B Fusobacterium nucleatum ATCC 25586= Gardnerella vaginalis ATCC 140BT Haemophilus influenzae NCTC 8143T Janthinobacterium lividum NCTC 9796T Legionella pneumophila ATCC 33153 Leptotrichia buccalis NCTC 10249T Moraxella lacunata NCTC 7911 Neisseria Pavescens ATCC 13120T Oceanospirillurn pusillum I F 0 13613T Oligella urethralis CCUG 994 Pseudomonas fluorescens ATCC 13525T Streptobacillus moniliformis NCTC 10651T Tissierella praeacuta ATCC 25539T Unidentified strain CCUG 10372

Other designations“

LMG 7S43T, Fennell 165T LMG 8559 LMG 8558 LMG 7546T, Fennell 231T LMG 7S39=, KO0456591 LMG 8773, CT1 LMG 8775, Pylo 10 LMG 8776, LMG 8928, NCTC 12031 LMG 8458, LMG 8738, CCUG 23435 LMG 8457 LMG 7608T, DSM 1740‘ LMG 7466T, CCUG 13145= LMG 7792, Skirrow 912/79 LMG 7793, DSM 806 LMG 8192 LMG 196 LMG 2370‘ LMG 4343T LMG 3816T LMG 8206T, ATCC 29798’ LMG 8202T

LMG 1340T LMG 2093 LMG 7832’r LMG 2892” LMG LMG LMG LMG LMG

1009, ATCC 17952 5297=, CCUG 34ST 5308= 5304 1794T

LMG 8203T LMG 7819, Skirrow 912/79

Received from”:

Source

Fennel1 Claesson Our own isolate Fennell Goodwin Mars hall Megrau d NCTC

Rectal swab of a homosexual male Blood of 42-yr-old female Feces of 1-yr-old female Rectal swab of a homosexual male Endoscopic biopsy specimen Duodenum Gastric mucosa Ferret gastric mucosa

ATCC

Patient with gastroenteritis

ATCC CCUG DSM Skirrow

Patient with gastroenteritis Bovine rumen fluid Bovine rumen fluid Rectal swab of a homosexual male

DSM Pfennig Mannheim ICPB ATCC ATCC CBRI ATCC Mannheim Mannheim ATCC Mannheim Mannheim Mann heim NCIB

Anaerobic sludge

Mannheim ATCC Mannheim Sneath Mannheim Mannheim NCTC ATCC IF0 Bevre MMCA Mannheim ATCC Skirrow

Bovine lesion Freshwater Air Perineum scar Human periodontal pocket Abdominal abscess Bovine Human blood Soil Cervicofacial lesion Human vaginal secretions Soil Human lung Supragingival calculus Human spinal fluid Mussel Prefilter waterworks tanks Human case of rat bite fever Gangrene 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. t l and t2 refer to different colony types.

Organisms belonging to rRNA cluster I Campylobacter fetus subsp. fetus CCUG 6823AT C. fetus subsp fetus ATCC 33246 C. fetus subsp. fetus NIDO 212514 C. fetus subsp. fetus NIDO 7572 C. fetus subsp. venerealis CCUG 53ST C. fetus supsp. venerealis NIDO 483 C. hyointestinalis CCUG 14169T C. hyointestinalis CCUG 24181 C. hyointestinalis CCUG 14916 C. hyointestinalis CCUG 20823 C. concisus CCUG 13144T C. concisus CCUG 20534 C. concisus CCUG 17580 C. concisus CCUG 18688 C. concisus CCUG 19219 C. mucosalis CCUG 6822T C. mucosalis CCUG 10771 C. mucosalis FS 921177 CLO strain CCUG 20705 C. sputorum biovar sputorum CCUG 972ST C. sputorum biovar bubulus CCUG 11289 C. sputorum biovar bubulus ATCC 33491 C. sputorum biovar fecalis CCUG 12015 C. sputorum biovar fecalis CCUG 12017 C. sputorum biovar fecalis CCUG 17695B C. coli CCUG 11283= C. coli CCUG 10369 C. jejuni subsp. jejuni ATCC 33250 C. jejuni subsp. jejuni CCUG 14914 C. jejuni subsp. jejuni RV4 C. jejuni subsp. jejuni CCUG 10370 C. jejuni subsp. doylei CCUG 18265 C. lari NCTC 11352T C. lari CCUG 12774 C. lari CCUG 18267 “C. upsaliensis” CCUG 14913” “C. upsaliensis” B523 “C. upsaliensis” E282 “C.upsaliensis” CCUG 20818 C. cuwus CCUG 13146T C. rectus CCUG 11640 [Wolinella] sp. strain CCUG 11641 [Bacteroides]gracilis FDC 404 [Bacteroides] ureolyticus CCUG 7319T

Source of DNA

29.5

37.9 38.4 38.6 39.5 40.4 37.1 36.1 37.4 34.2 30.8 29.9 29.8 30.5 31.8 31.8 30.8 32.5 30.0 32.6 30.9 30.1 30.5 30.4 31.8 30.5 33.5 32.8 32.8 35.4 45.1 45.4 45.7

33.9 33.7 34.5 34.3 33.2 33.9 35.1 33.6 35.2

G+C content (mol%)

0.23 0.17 0.22 0.28 0.18 0.16 0.12 0.15 0.17 0.15 0.13 0.13 0.14 0.11 0.11 0.14 0.10 0.13 0.16 0.14 0.12 0.15 0.15 0.10 0.14

0.15 0.11 0.10 0.10 0.15

76.6 76.4 76.9 76.5 76.6 76.3 73.5 74.8 76.6 74.7 70.9 72.3 71.8 72.3 71.4 70.7 70.9 70.2 70.7 71.5 70.6 69.4 70.7 69.5 70.0

69.2 68.0 71.0 71.5 68.9

fetus subsp. CCUG 6823AT

0.19

71.8

69.4 71.6 72.2

68.5

0.13 0.13 0.13

0.10 0.15 0.16 0.11 0.11 0.12 0.10 0.11 0.10 0.10 0.19

70.0 69.4 69.1 68.3 66.9 70.7 71.4 68.2 69.6 70.0

0.17 0.15 0.12 0.15 0.10

0.11 0.10 0.12 0.11 0.20 0.12 0.19 0.23 0.21 0.16 0.14

71.2 71.6 70.4 70.1 76.9 76.5 76.5 76.9 77.1 76.1 71.8

0.18 0.20 0.19 0.12 0.25 0.15 0.14 0.16 0.15

76.0 77.3 76.3 75.2 75.5 74.9 73.4 73.5 72.3

71.5 70.7 70.3 70.9 67.1

0.12 0.13 0.14

70.7 71.5 71.4

0.21

71.8

0.11

0.17

71.7

0.16

71.3

69.9

0.16 0.13 0.22 0.17 0.15

~

72.1 71.3 71.7 71.5 71.1

$

0.22 0.17

b

~

CamW1obacter sputorum

71.7 71.8

concisus CCUG 20534

Camwlobacter Campylobacter

0.23 0.16 0.25 0.19 0.18 0.20 0.16 0.19 0.20 0.18 0.17 0.15 0.14 0.17 0.09 0.11 0.10 0.19

77.1 76.2 76.6 76.3 76.3 75.8 77.1 73.1 74.7 75.1 73.3 73.6 72.5 72.0 67.2 67.7 67.1 67.4

0.09 0.16 0.19

0.13

70.2

69.7 70.0 70.4

0.15 0.14

0.12

0.12

0.17 0.16 0.16

69.2 69.1

70.3

67.9

69.3 70.7 70.3

bacter coli CCUG ~ ~ 11283T ~ ~

campyl0-

64.6

68.2

66.6

0.09

0.09

0.11

Arcobacter nitrofigilis CCUG 12022 $

Hybridized with rRNA from:

62.4

63.8

63.6

65.8

64.9

0.18

0.29

0.35

0.26

0.13

CCUG 10373

62.2 62.9 62.7

62.5

61.8

63.7

63.4

64.1

0.06 0.10 0.08

0.07

0.09

0.13

0.08

0.09

CCUG 1314ST

Wolinella

TABLE 2. DNA base compositions and parameters of the DNA-rRNA hybrids formed by using labeled 23s rRNAs from Campylobacter, Arcobacter, and Wolinella strains

r

0

3E

p

v1

v)

U

N W

Reference strain of Sandstedt et al. (47). Data from reference 27. Data from reference 65.

Organisms belonging to rRNA cluster I1 Arcobacter nitrofigilis CCUG 15893T A. nitrofigdis CCUG 12022 A . cryaerophilus CCUG 17801T A. cryaerophilus CCUG 17805 A. cryaerophilus CCUG 12018 CLO strain CCUG 10373 CLO strain CCUG 10374 CLO strain CCUG 10375 Organisms belonging to rRNA cluster I11 Helicobacter cinaedi CCUG 188BT H. cinaedi CCUG 15432 H. cinaedi CCUG 17733 H. fennelliae CCUG 18820T H. pylon’ CCUG 17874T H. pylon CCUG 19106 H. mustelae CCUG 23652 CL03 strain CCUG 14564 “Flexispira rappini” ATCC 43879 “F. rappini” ATCC 43880 Wolinella succinogenes CCUG 13145T W succinogenes DSM 1740T Other campylobacters and reference strains CLO strain CCUG 13942 [Spirillum]sp. strain 5175 Aquaspinllurn serpens ATCC 1263gT Bacteroides coagulans CCUG 10974T Bacteroides fiagilis NCTC 9345 Bacteroides oralis ATCC 33269T Bacteroides splanchnicus ATCC 29572T Brucella melitensis NCTC 10094T Fusobacten’umnucleaturn ATCC 25586T Gardnerella vaginalis ATCC 1401gT Haemophilus influenzae NCTC 8143T Legionella pneurnophila ATCC 33153 Leptotnchia buccalis NCTC 10249T Oceanospirillum pusillurn IF0 13613T Streptobacillus moniliformis NCTC 10651T Tissierella praeacuta ATCC 25539T Unidentified strain CCUG 10372 51.0 24.2 29.3 58.5

42.0 38.9 37.8

43.1 57.9

41.6’ 38.4‘ 50.0 36.3

36.1 37.3 38.3 34.9 35.6 36.8 42.2 47.4 33.7 34.3 47.2 47.0

29.0 28.5 28.6 28.6 28.4 28.0 29.2 29.4

52.4

0.09

0.04 0.05 0.03 0.12 0.03

0.04

52.7

58.8 52.0 50.2 57.8 58.7

0.09

0.12

64.4

54.0

0.08

0.11

68.5

62.2

0.09

66.6

64.1 64.4

0.05 0.04

55.7

53.8 58.8

52.5

0.10

0.05 0.08

0.03

0.05 0.16

0.06 0.06

65.3 65.1 53.6 47.3

0.13

65.8

0.04 0.05 0.06

0.04 0.06 0.06 0.07 0.09

64.4 64.0 63.3 62.8 62.7 63.5 63.4 63.6

0.07 0.09 0.13 0.06 0.12 0.15

64.9 65.7 66.2 67.8 66.8 66.0

63.1 64.3

63.2

67.9

65.9

65.2

0.05 0.04

0.08

0.15

0.12

0.06

67.0

64.2

76.3 76.7 71.8 73.2 72.3 73.8 70.1 72.4

0.13

0.12

0.11 0.17 0.17 0.17 0.17 0.20 0.17 0.18

51.8

62.4

72.7 73.3 72.6 74.1 73.4 77.4 71.9 71.4

0.09

0.14

0.14 0.16 0.20 0.18 0.26 0.34 0.18 0.19

63.7 64.9

71.2 71.9 71.2 71.1 68.5 69.6 71.3 69.8 70.5 71.3 79.1 78.4

65.7

65.6

64.5 64.2

0.05 0.07

0.05 0.08 0.06 0.09 0.11 0.10 0.10 0.05 0.07 0.08 0.20 0.18

0.18

0.14

0.07 0.08

w W

c,

2

F5

8

v1

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 DNA from:

Labeled rRNA from:

TmW

("C)

% of rRNA binding

Campylobacter fetus subsp. fetus CCUG 6823AT C. fetus subsp. fetus NIDO 7572 C . fetus subsp. fetus NIDO 7572 C . fetus subsp. fetus NIDO 7572 C. fetus subsp. fetus NIDO 7572 C. feius subsp. .fetus NIDO 7572 C. fetus subsp. fetus NIDO 212514 C. fetus subsp. fetus NIDO 212514 C. fetus subsp. fetus NIDO 212514 C . fetus subsp. venerealis CCUG 7477 C. fetus B5 C . fetus B5 C. fetus B5 C. jejuni subsp. jejuni ATCC 33250 C. jejuni subsp. jejuni M2 C. jejuni subsp. jejuni M2 C. jejuni subsp. jejuni M2 C. jejuni subsp. jejuni M2 C. jejuni subsp. jejuni JJ91 C. jejuni subsp. jejuni CCUG 10370 C. sputorum biovar bubulus ATCC 33491 C. sputorum biovar bubulus ATCC 33491 C. sputorum biovar bubulus ATCC 33491 Arcobacter nitroJgilis CCUG 12022

Cardiobacterium hominis ATCC 15826T Cardiobacterium hominis ATCC 15826T Cytophaga johnsonae ATCC 17061T Oligella urethralis CCUG 994 Brucella abortus ATCC 2344gT Neisseria javescens ATCC 13120T Cardiobacterium hominis ATCC 15826* Moraxella lacunata ATCC 17952 Actinobucillus lignieresii NCTC 4189T Cardiobacterium hominis ATCC 15826T Janthinobacterium lividum NCTC 9796Ta Arthrobacter oxydans CBRI 21010T Escherichiu coli B Curdiobacterium hominis ATCC 15826T Pseudomonas Jluorescens ATCC 13525=' Janthinobacterium lividum NCTC 9796T" Agrobacterium tumefaciens ICPB TTlll Escherichia coli B Pseudomonas Jluorescens ATCC 13525Tb Cardiobacterium hominis ATCC 15826T Cardiobacterium hominis ATCC 15826T Aciinobacillus lignieresii NCTC 4189T Neisseria Jlavescens ATCC 13120T Aquaspirillum ayuaticum ATCC 11330T

60.1 58.1 55.3 52.4 56.9 53.8 53.2 55.7 57.2 57.4 55 .O 58.0 59.5 60.1 55.0 57.0 57.0 58.0 54.0 59.7 58.8 57.5 52.2 51.6

0.07 0.07 0.04 0.05 0.08 0.08 0.07 0.06 0.05 0.05 0.12 0.14 0.08 0.07 0.10 0.10 0.07 0.06 0.07 0.06 0.06 0.04 0.05 0.09

(I

Data from reference 12.

' Data from reference 13.

tively labeled rRNAs from strains belonging to gram-negative reference taxa. Table 2 shows the results of DNA-rRNA hybridizations between DNAs from campylobacters, related taxa, and gram-negative reference strains and radioactive rRN As from Campyfobacter, Arcobacter, and Wolinelln strains. The DNA-rRNA hybridization results are presented as a dendrogram based on the T,(,, values of the hybrids in Fig. 1. The DNA-rRNA hybridizations between DNAs from campylobacters and related strains and rRNAs from gramnegative reference strains belonging to rRNA superfamilies I to V (Tables 2 and 3) revealed Tmcc)values between 50.2 and 60.4"C (average, 56.6 2 3.1"C). The DNA-rRNA hybridization results show that within rRNA superfamily VI, three major rRNA homology groups, called rRNA clusters I, 11, and 111, can be differentiated (Fig. 1).rRNA cluster I is linked to rRNA cluster 11 at an average 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 structure of these rRNA clusters is shown in Fig. 1. rRNA cluster I contains Campylobacter fetus subsp. fetus (four strains), Campylobacter fetus subsp. venerealis (two strains), Campylobacter hyointestinalis (four strains), Campylobacter concisus (five strains), Campylobacter mucosalis (three strains), Campylobacter sputorum (six strains representing Campylobacter sputorum biovar sputorum, Campylobacter sputorum biovar bubulus, and Campylobacter sputorum biovar fecalis), Campylobacter coli (two strains), Campylobacterjejuni subsp. jejuni (four strains), Campylobacter jejuni subsp. doylei (one strain), Campylobacter lari (two strains), "Campylobacter upsaliensis" (four strains), Campylobacter curvus (one strain), Campylobacter rectus (one strain), and the generically misnamed organisms [Bacteroides] gracilis (one strain) and [Bacteroides] ureolyticus (one strain).

The following three strains also belong to rRNA cluster I: CLO strain CCUG 20705, [Wolinella]sp. strain CCUG 11641 (57), and strain CCUG 18267. The latter strain was received as a representative of the urease-positive therrnophilic Campylobacter group (3,4). This group of thermophilic campylobacters was identified as Campylobacter lari (38) by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. We performed DNA-DNA hybridizations between this strain and Campylobacter lari CCUG 12774 and found a level of DNA binding of 67% (data not shown). When DNA-DNA hybridizations were performed between DNA from strain CCUG 20705 and DNAs from Campylobacter fetus, Campylobacter hyointestinalis, Campylobacter concisus, and Campylobacter mucosalis strains, no significant DNA binding values were detected (data not shown). rRNA cluster I1 consists of Arcobacter nitrojigilis (two strains), Arcobacter cryaerophilus (three strains), and CLO strains CCUG 10373, CCUG 10374, and CCUG 10375. rRNA cluster 111 consists of Wolinella succinogenes (two strains), Helicobacter pylori (two strains), Helicobacter mustelae (one strain), Helicobacter cinaedi (three strains), Helicobacter fennelliae (one strain), "Flexispira rappini" (one strain), and strain CLO-3 of Fennel1 et al. (16). The free-living campylobacters of Laanbroek et al. (27) and Wolfe and Pfennig (65) do not belong to one of the three major rRNA homology groups. They have a separate position on the T,,z(c)dendrogram (Fig. 1) at an average T,(,, of 643°C (Table 2). DNA-DNA hybridization results. DNA-DNA hybridizations were performed with nine strains belonging to rRNA cluster 11 (Fig. 2). Our hybridization results revealed the presence of three DNA homology groups and one separate strain (CLO strain CCUG 10373). Within the groups, the DNA binding values varied from 46 to 100%. Significant

VOL. 41, 1991

TAXONOMY OF rRNA SUPERFAMILY VI

95

Campylobacter concisus

----

Y I I I

Campylobacter curvus rectus [Wolinella] sp. CCUG 11641

I Campylobacter

I

I

Campylobacter mucosalis CLO strain CCUG 20705 Campylobacter sputorum

- -“Campylobacter

rRNA cluster I

upsaliensir”

7 +I Campylobactern i!;[ I-

Campylobacter lari

i [Bacteroides] ureolyticus I [Bacteroides]

gracilis

Arcobacter nitrofigilis I.

I

I.

CLO strain CCUG 10373

--- Arcobacter cryaerophilus

[

CLO strains CCUG 10374

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 CCUG 10374 and Arcobacter cryaerophilus strains. Immunotyping analysis. Immunizing of rabbits was started in January 1980. We prepared 130 soluble antigens and 34 antisera, representing all of the taxa belonging to rRNA superfamily VI. We prepared antigens from at least the type strain of each taxon. The numbers of strains tested per taxon and the average precipitation values versus each antiserum are shown in Table 4. A total of 16 major groups, 6 of which could be further subdivided, were delineated (Table 4). Significant cross-reactions (values of 1 3 ) were observed only between members of the same rRNA branch. Immunotyping group (ITG) 1 contained Campylobacter fetus and Campylobacter hyointestinalis. Campylobacterfetus subsp. fetus and Campylobacter fetus subsp. venerealis formed subgroup l a , and Campylobacter hyointestinalis constituted subgroup l b . Campylobacter concisus, Campylobacter mucosalis, and CLO strain CCUG 20705 constituted ITG 2 (subgroups 2a through 2c, respectively). ITG 3 contained the organisms of the Campylobacter sputorum rRNA branch (Campylobacter sputorum biovar sputorum, Campylobacter sputorum biovar bubulus, and Campylobacter sputorum

biovar fecalis); all Campylobacter sputorum strains gave similar precipitation values against the three antisera. ITG 4 contained Campylobacter curvus and Campylobacter rectus (subgroups 4a and 4b, respectively). ITG 5 contained all of the taxa belonging to the Campylobacter coli rRNA branch, including Campylobacter coli, Campylobacter jejuni subsp. jejuni, Campylobacter jejuni subsp. doylei, Campylobacter lari, and “Campylobacter upsaliensis,” each of which constituted a separate subgroup (subgroups 5a through 5e, respectively). ITGs 6, 7, and 8 each contained a single taxon ([Bacteroides]gracilis, [Bacteroides] ureolyticus, and Arcobacter nitrofigilis, respectively). Arcobacter cryaerophilus and CLO strains CCUG 10373 and CCUG 10374 constituted ITG 9. Arcobacter cryaerophilus and CLO strain CCUG 10374 were immunologically similar (subgroup 9a); CLO strain CCUG 10373 (subgroup 9b) gave weaker, but still clear-cut cross-reactions versus Arcobacter cryaerophilus and CLO strain CCUG 10374 (Table 4). ITG 10 contained only Wolinella succinogenes. ITG 11 consisted of Helicobacter cinaedi (subgroup 1l a ) and Helicobacter fennelliae (subgroup l l b ) . Helicobacter pylori, Helicobacter mustelae, “Flexispira rappini,” strain CLO-3, and the Campylobacter

4

5 3

5a 5b SC 5d 5e

7 14 3 3 6

2

2

3

4

C. coli C. jeuni subsp. jejuni C. jejuni subsp. doylei C. lari "C. upsaliensis"

[Bacteroides]ureolyticus

[Bacteroides]gracilis

Arcobacter nitrofigilis

A. cryaerophilus CLO strain CCUG 10373

lla llb

12

8

7

2

Helicobacter cinaedi H. fennelliae CCUG 18820T

H. pylon

H. mustelae

15

16

CLO-3 strain CCUG 14564

CLO strain CCUG 13952 1

0

0

0

0 0

0

0 0

0

0

0

0 0 0 0 0

0 0

1 0

0

-

0

0

-

0

0

0 0

0

0 0

0

0

0

1 1 1 1 0

0 0

0 0

0

0 1 0

7 6

8

-

2

0

0

1

0 0

0

1 0

0

0

0

1 2 2 2 2

3 2

2 1

1

3 3 3

6 7

5

-

0

0

0

0

0 0

0

0 0

0

0

0

0 0 0 1 0

1 1

0 0

0

7 2 2

0 0

0

0

2

0

-

1

2

2

0

0

0 0

0

0

0

0

0

0

0

0 0

0

0 0

1 0

0

2 -

1

1

0

3

1 0

0 0

1 2

8 8

8

1 1 2

0 0

0

2 2 2 3 3

3 2

3 2

3

6 6 8

1 2

1

0

0

0

0 1

0

0 0

0

0

1

0 0 0 1 0

3 2

1 0

1

5 8 2

2 2

0

0

0

0 0

0

0 0

0

0

0

0 0 0 1 0

2 0

0 0

0

8 0 0

0 0

0

0

0

0

0

0 0

1

0 0

0

0

0

1 2 2 1 0

1 0

7 7

8

1 0 0

0 0

0

0

-

-

0

0

0

0 0

0

0 0

0

0

0

0 0 0 0 0

0 0

4 3

3

0 0 0

0 0

0

0

0

2

0

0 0 2

7 6 6 5 6

0

-

-

0

0

0

0 1

0

-

-

0

0

0

0 0

0

- - 2 - - 2

0

2

1

0

0

0

1 0

1

0

2 1 0

1

2 2 1 3 3

7 2 2 7 8 2

1 0 2 2 0 2

0

0 0 0 2 1 1 1 0 2

0 0 0 0 0 0

0

0

0

0

0

0

0 0

0

0 0

0

0

0

5 6 6 2 3

0 0

0 0

0

0 0 0

0 0

0

0

-

0

0

1

0 1

1

0 0

0

0

1

4 4 7 2 3

1 1

0 0

1

0 0 1

0 0

0

0

0

0 0

1

1

1

0

0

0

0 0

0

1 -

0

0

0 -

0 -

0

0

0

0 0

0

0 0

0

0

0

2 2 1 4 2 3 4 7 1 4

0 1

0 0

0

0 0 0 0 0 0

0 0 0 0

0

3

0

0

0

0

0 0

0

0 0

0

0

0

3 2 2 2 5

0 0

1 0

0

0 0 0

0 0

0

1 1 1

2 2 2

0 0 0 0

0

1 3 1 4

8

4 1

7

-

0

1

1

2 0

0

1

1

2

1

3 2

1

1 1 1 -

1

1

0 - 0

-

0

0

0

0 0

0

0 0

0

0

8

0 2 2 1 2 2 1 2 1 1 1 2 1 1 3

0 0

2 3 3 1 2 2

0 - 3

0 0 0

0 0

0

3

0

1

0

1

0 0

0

2 0

6

0

0

0 0 0 0 0

0 0

1 0

0

0 0 0

0 0

0

0

0

0

0

0

0 0

0

6 0

0

0

0

0 0 0 0 0

0 0

0 0

0

0 0 0

0 0

0

2

0

0

0

0

0 0

1

6 3

1

0

0

0 0 0 0 0

0 0

0 1

0

0 0 1

0 0

0

0

0 0

0

0 0

0

0 0

0

0 0

0

0 0

0

0 0

0

2

0

0

0

0

0

0

0 0 0

0 0

0

0

0 0

0

0

0 0 0 0 0 0

0 0

0

0

1

0 0 0 0 0

0 0

0

0

0

2 1 1 2 2

2 1

0

0

0

0 1 2 0 0

0 0

1 0

0 0

0 0

0 0

0 0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0 0 0 0 0 0 0 0 0 0 0 0 1 - 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0

0

0

0

0

0

0 0

0

0

0

0

0

0

0 0

0

0

2

0

3

0

2 2

6

0

2

0

0

0

6 3

0

0

1

0

0

0

3 0

0

-

0

6

3

1

2 1

0

0

0

0

2

6

0 0

0

0

0

0

3

7

0 0

0

0

6

0

0

0

0 2

0

5

0

0

0

0

0 0

0

8 3 2 1 0 - 0 0 0 0 4 7 - 0 0 - 0 0 0 0

2

0

0

0 0 1 0 0

0 0

0 0 2 0 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0 1

1

0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 1 0 0 0 0

0 0

0

8

13 3

a 0, No precipitate; 1or 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.

1

"Flexispira rappini" ATCC 43879

13

10

Wolinella succinogenes CCUG 13145"

9a 9b

8

7

6

4a 4b

3 3

3

C. curvus CCUG 13146" C. rectus CCUG 20446"

CLO strain CCUG 20705

Camwlobacter sputorum biovar sputorum CCUG 9728" C. sputorum biovar bubulus C. sputorum biovar fecalis

2a 2b 2c

0 1 0

18 11

C. concisus C. mucosalis

lb

8 8 5

la

la

9

ITG

5 8

No. of strains tested

Camwlobacterfetus subsp. fern C. fetus subsp. veneredis C. hyointestinalis

Antigen(s) from:

~

Reaction with antiserum againsta:

~~

TABLE 4. Results of immunotyping analysis

VOL.41, 1991

97

TAXONOMY OF rRNA SUPERFAMILY VI TABLE 5 . Characteristics that differentiate species within the genus Arcobacter

Taxon

A rcobacter nitrojigilis' Arcobacter cryaerophilus' CLO strains CCUG 10374 CCUG 10375 CCUG 10373

Urease

Nitro-

activity

activity genase of nitrites

Reduction Hydrolysis of indoxyl acetate

Hydrogen

Susceptibility to:

Anaerobic growth Aerobic without

production" Nalidixic Cepha- growth acidb lothinb

aspartate and furnarate

Growth in the

Colony

presence of 3.5% NaCl

Swarming on moist media

Vd

+

-

-

+

S

S

-

-

+

White

-

-

-

+

+

-

V

R

+

+

-

Yellow or colorless

+

-

ND ND ND

ND ND ND

+ +

-

S S S

S S R

+ + +

-

Colorless Colorless 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 ITG (ITGs 12 through 16, respectively). Phenotypic tests. The results of the phenotypic tests performed with CLO strains CCUG 10373, CCUG 10374, and CCUG 10375 are included in Table 5 and in the description of the genus Arcobacter given below. DISCUSSION In previous papers by workers from our laboratory, the significance and applications of the DNA-rRNA hybridization technique have been established (12-14, 45, 46, 63). In the last few years, DNA-rRNA hybridization results have been confirmed by rapidly accumulating 16s rRNA sequencing data (64). On the basis of T,(,, values, a phylogenetic tree of the major part of the gram-negative bacteria has been constructed (9, 13). Within this phylogenetic tree, it is possible to distinguish at least five major groups, called rRNA superfamilies, which are related only above the family level (9). Preliminary DNA-rRNA hybridization results (60) indicated that the genus Campylobacter does not belong to one of the five rRNA superfamilies. The purposes of this study were (i) to determine the taxonomic position of the genus Campylobacter; (ii) to unravel its genotypic structure; and (iii) to determine the phylogenetic relationships among the genera Campylobacter, Wolinella, Helicobacter, and "Flexispira" and related organisms. Therefore, we prepared seven radioactive labeled rRNAs, more than 70 DNAs, 34 antisera, and 130 antigens from campylobacters and related organisms. Taxonomic position of the genus Campylobacter. The DNArRNA hybridizations between Campylobacter strains and gram-negative reference strains belonging to rRNA superfamilies I to V (Tables 2 and 3) revealed an average T,(,, value of 56.6 5 3.1"C. This confirms and extends our preliminary results (60), namely, that the genus Campylobacter and its relatives do not belong to one of the five previously described rRNA superfamilies (sensu De Ley [9]) within the gram-negative bacterial group, but constitute the core of a sixth rRNA superfamily. The value 56.6"C is the lowest average linking level between two rRNA superfamilies; it indicates that the genus Campylobacter and related taxa are phylogenetically far removed from the other gram-

negative bacteria. These organisms definitely belong to a separate eubacterial phylum, as suggested by Romaniuk et al. (42). Taxonomic structure of the genus Campylobacter and relationships with other bacteria belonging to rRNA superfamily M. In agreement with the results of Paster and Dewhirst (39) and Thompson et al. (58), we found an extremely high level of phylogenetic heterogeneity within the previously defined genus Campylobacter. This heterogeneity can be compared with the heterogeneity within the largely generically misnamed genus Pseudomonas (13). Organisms belonging to different rRNA homology groups of the genus Pseudomonas have AT,,,, values of up to 10 to W C , values which are similar to the AT,,,, values for the organisms belonging to the three major rRNA homology groups within rRNA superfamily VI (Fig. 1). Cluster analysis of the T,(,) values revealed the presence of three major rRNA clusters (Fig. 1). The compositions of these rRNA clusters entirely coincide with the compositions of the rRNA homology groups inferred from 16s rRNA sequence analysis (39, 58); only their branching order is reversed. These high AT,,,, values (approximately 10 to W C ) undoubtedly reflect relationships at or above the genus level (11,13). Therefore, the taxonomy of the former genus Campylobacter and related taxa should be revised. rRNA cluster I. The rRNA branches within rRNA cluster I are linked at AT,,,, values of about 6 to 8°C (Fig. 1).Within each rRNA branch, all of the taxa have significant precipitation values, which reveal immunological similarities within each rRNA branch (Table 4). Differences in T,(,) values of 6 to 8°C could point to intergeneric relationships provided sufficient phenotypic arguments are available. However, as Paster and Dewhirst have noted (39), there are at present few known characteristics which separate the taxa belonging to rRNA cluster I. It is striking that all thermophilic (or thermotolerant), enteropathogenic Campylobacter species belong to the Campylobacter coli rRNA branch (Fig. 1).The Campylobacter sputorum rRNA branch contains organisms that are commensal for humans and animals, while the Campyfobacter fetus and Campylobacter concisus rRNA branches contain organisms that are pathogenic or associated with diseases, mainly in animals. The former Wolinella strains (Campylobacter curvus, Campylobacter rectus, and [ Wolinella] sp. strain CCUG 11641), [Bacteroides] gracilis,

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VANDAMME ET AL.

and [Bacteroides] ureolyticus are found in oral cavities in higher numbers in patients with periodontal disease than in healthy individuals. However, there are so far no phenotypic characteristics that are useful for differentiating the groups of taxa belonging to each rRNA branch. On the contrary, all Campylobacter and former Wolinella strains belonging to rRNA cluster I share a considerable number of phenotypic features (see below). Furthermore, they have similar isoprenoid quinone compositions (35, 36), which is a taxonomically valuable feature (6). In contrast to most other gramnegative bacteria, campylobacters do not contain ubiquinones as major isoprenoid quinones but do contain menaquinones. All of the Campylobacter strains belonging to rRNA cluster I (including Campylobacter curvus and Campylobucter rectus) that were tested have menaquinone 6 and methyl-substituted menaquinone 6 as their major isoprenoid quinones (35, 36). Studies of the cellular fatty acid compositions of these organisms revealed that they all have the same major fatty acids (i.e., tetradecanoic acid, hexadecanoic acid, hexadecenoic acid, and octadecenoic acid [21, 281) (data for Campylobacter curvus and Campylobacter rectus were obtained from the database of the Microbial Identification System described by Miller [33]). Most campylobacters have DNA base ratios in the range from 30 to 41 mol% G + C , while Campylobacter curvus, Campylobacter rectus, and [Wolinella] sp. strain CCUG 11641 have G+C contents between 45 and 46 mol% (Table 2). This G+C content range is rather large but not unique in bacterial taxonomy (39). On the basis of the numerous genotypic and phenotypic similarities, we conclude that the genus Campylobacter should be restricted to the Campylobacter species belonging to rRNA cluster I and that the former species [ Wolinella] curva and [Wolinella] recta and [Wolinella] sp. strain CCUG 11641 should be included in the genus Campylobacter as Campylobacter curvus, Campylobacter rectus, and Campylobacter sp. incertae sedis, respectively. Tanner and co-workers found no significant levels of DNA homology between Campylobacter curvus and Campylobacter rectus and between Campylobacter curvus and Campylobacter sp. strain CCUG 11641; DNA homology values ranging from 34 to 55% were found between Campylobacter rectus and Campylobacter sp. strain CCUG 11641 (54, 55, 57). Campylobacter curvus and Campylobacter rectus are immunologically more closely related to each other than to the other Campylobacter taxa (Table 4). Emendation of the genus Campylobacter. In Bergey ’s Manual of Systematic Bacteriology, the genus Campylobacter consists of the following five species: Campylobacterfetus, Campylobacter jejuni, Campylobacter coli, Campylobacter sputorum, and Campylobacter concisus (48). DNA-DNA hybridization studies showed that Campylobacter sputorum subsp. mucosalis represents a separate species (i.e., Campylobacter mucosalis) and that Campylobacter sputorum subsp. sputorum, Campylobacter sputorum subsp. bubulus, and “Campylobacter fecalis” (17) should be considered biovars of Campylobacter sputorum (43, 44). Our DNArRNA hybridization results confirm that Campylobacter lari, Campylobacter hyointestinalis, and “Campylobacter upsaliensis” are true Campylobacter species (29, 39, 42, 58). Recently, two groups of CLO strains, the nitrate-negative Campylobacter strains (25, 50) and the urease-positive thermophilic Campylobacter strains (3, 4), were shown to be atypical Campylobacter jejuni and Campylobacter lari strains, respectively (38, 51). The name Campylobacter jejuni subsp. doylei was given to the nitrate-negative Campylobacter strains (51). Several of these recently described

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SYST. BACTERIOL.

Campylobacter taxa have characteristics that do not correspond to the characteristics given in the description of the genus Campylobacter in Bergey ’s Manual of Systematic Bacteriology (48). Therefore, below we propose an emended description of the genus Campylobacter. Emended description of the genus Campylobacter Sebald and Veron 1963. Slender, spirally curved, gram-negative rods that are 0.2 to 0.5 pm wide and 0.5 to 5 pm long. The rods may have one or more spirals and can be as long as 8 pm. They also appear to be S shaped and gull winged when two cells form short chains. Nonsporeforming. Cells in old cultures may form spherical or coccoid bodies. Motile (with a characteristic corkscrewlike, darting motion) by means of a single polar unsheathed flagellum at one or both ends of the cell. Microaerophilic to anaerobic with a respiratory type of metabolism. Growth at 37°C; no growth at 15°C. An 0, concentration of 3 to 15% is required for the preferentially microaerophilic species, while most strains (Campylobacter curvus and Campylobacter rectus) which prefer to grow under anaerobic conditions can also grow in the presence of 1to 5% 0,. Hydrogen is required or stimulates growth under both microaerophilic and anaerobic conditions. Occasionally a few strains may grow slightly under aerobic conditions (20% 0,). Chemoorganotrophs. Carbohydrates are neither oxidized nor fermented. Does not require serum or blood for growth. Energy is obtained from amino acids or tricarboxylic acid cycle intermediates and not from carbohydrates. Urea (except for a group of atypical Campylobacter lari strains [4])and gelatin are not hydrolyzed. Oxidase activity, but no lipase activity. No pigment production, except for Campylobacter mucosalis and Campylobacter hyointestinalis, which produce a dirty yellow pigment. Menaquinone 6 and methyl-substituted menaquinone 6 are the major respiratory quinones ; tetradecanoic acid, hexadecanoic acid, hexadecenoic acid, and octadecenoic acid are the major fatty acids. The G + C content of the DNA ranges from 30 to 46 mol%. Several species are pathogenic for humans and animals. Found in the reproductive organs, intestinal tracts, and oral cavities of humans and animals. The type species is Campylobacter fetus (Smith and Taylor 1919) Sebald and Veron 1963. This emended genus contains the following species: Campylobacter fetus, Campylobacter hyointestinalis, Campylobacter mucosalis, Campylobacter concisus, Campylobacter sputorum, Campylobacterjejuni, Campylobacter coli, Campylobacter lari, “Campylobacter upsaliensis,” Campylobacter curvus, and Campylobacter rectus. All other Campylobacter species are generically misnamed. Description of Campylobacter curvus comb. nov. Campylobacter curvus (basonym, Wolinella curva Tanner, Listgarten, and Ebersole 1984) (curv’us. L. adj. curvus, curved). The description is the same as that given previously for [ Wolinella] curva (55). Description of Campylobacter rectus comb. nov. CampyLobacter rectus (basonym, Wolinella recta Tanner, Badger, Lai, Listgarten, Visconti, and Socransky 1981) (rect’us. L. adj. rectus, straight). The description is the same as that given previously for [ Wolinella] recta (54, 56). Taxonomic position of generically misnamed Bacteroides species. The taxonomic position of [Bacteroides]gracilis and [Bacteroides] ureolyticus remains uncertain. These organisms share a number of phenotypic characteristics with and have DNA base ratios in the range proposed for the emended genus Campylobacter (Table 2) (24, 39, 54). The fatty acid composition of [Bacteroides] gracilis resembles the fatty

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

Major isoprenoid quinones

Presence of hexadecanoic acid as a major component

MK-6, Un-MK-6

+b

Growth at: Genus

Nitrate Growth Hydrolreduc- on 0.5% ysis of tion glycine urea

Cell morpholOgY

No. of flagella

Position of Flagellar G + C flagella sheaths content (mol%)

15°C 30°C 42°C

Arcobacter

+‘

ND

V

+

+

-

Campylobacter‘

+d

V

-e

-

+

V

Wolinelld Helicobactefl

+

-

-

V

-

-

V

V

W V

“Flexispira”

-

+

-

-

+

+ +

Curvedand 1 spiral rods Curved and 1 spiral rods Spiral 1 Curved and V spiral rods Fusiform Multiple rods

Polar

Absent

28-31

Polar

Absent

30-46 MK-6, *MK-6

Polar Absent Polar and Present lateral Polar Present

47 MK-6, *MK-6 35-44 MK-6, Un-MK-6

33

ND

+ + -

ND

‘‘ 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; [Bacteroides] ureolyticus has a considerably different fatty acid composition, with hexadecenoic acid, 3-hydroxydecanoic acid, octadecanoic acid, and octadecenoic acid as the major fatty acids (data are from the database of the Microbial Identification System, as described by Miller [33]). Our DNA-rRNA hybridization results confirm that these organisms are closely related to the campylobacters (39), but additional data are required before definite conclusions can be drawn. rRNA cluster 11. rRNA cluster I1 is a rather homogeneous and clearly separated rRNA cluster (Fig. 1). We propose that the organisms in this cluster should be included in a new genus, Arcobacter, with two species, Arcobacter nitrojigilis and Arcobacter cryaerophilus. These two species and CLO strains CCUG 10373, CCUG 10374, and CCUG 10375 have similar G + C values (28 to 30 mol%) (Table 2) and share a considerable number of phenotypic characteristics (see below). The genus Arcobacter can be differentiated on the basis of phenotypic characteristics from the other genera of rRNA superfamily VI (Table 6). Arcobucter nitrofigilis and Arcobacter cryaerophilus can be differentiated from each other by their Tm(e, values versus reference rRNAs from

CCUG

No.

FIG. 2. DNA-DNA hybridization results for nine Arcobacter strains. Each DNA-DNA hybridization value is the average degree of binding from at least two experiments. Three DNA homology groups (with DNA bindig values of more than 46%) and one separate strain are clearly delineated.

members of rRNA cluster I1 (Table 2) and by their phenotypic (Table 5 ) and immunological (Table 4) characteristics. No significant DNA binding values were measured between Arcobacter nitrojigilis and Arcobacter cryaerophilus (Fig. 2). The fact that two species of one genus have such diverse habitats (Arcobacter cryaerophilus occurs in animal and human hosts [37], whereas Arcobacter nitrojigilis is a nitrogen-fixing, plant-associated bacterium [32]) is not unique in bacterial taxonomy. Similar situations occur in several other genotypically coherent genera (e.g., in the genera Xanthornanas [52] and Cornarnonas [53]). Strain CCUG 10373 has a separate position on the Tm(e)dendrogram (Fig. 1) and exhibited no significant DNA binding with the other organisms belonging to the genus Arcobacter (Fig. 2 ) . Strain CCUG 10373 is immunologically (Table 4) and phenotypically (Table 5 ) different from Arcobacter nitrojigilis, Arcobacter cryuerophilus and CLO strains CCUG 10374 and CCUG 10375. Additional strains should be isolated and studied before an appropriate species description can be proposed. We believe that CLO strain CCUG 10373 is an Arcobacter strain. CLO strains CCUG 10374 and CCUG 10375 are closely related to each other (96% DNA binding) (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 cryaerophilus strains (Table 2 and Fig. l ) , these two strains 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 et al. (32), Moss et al. (36), and Han et a]. (23). Description of Arcobacter gen. nov. Arcobacter (Ar’co. bac.ter. L. n. arcus, bow; Gr. n. bacter, rod; M. L. masc. n. Arcobacter, bow-shaped rod) cells are grarn-negative nonsporeforming rods (width, 0.2 to 0.9 pm; length, 1 to 3 Fm)

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that are usually curved, S shaped, or helical. Motile (with a darting, corkscrewlike motion) by means of a single polar, unsheathed flagellum. Growth occurs at 15,30, and 37°C; no growth occurs at 42°C. Optimal growth occurs under microaerophilic conditions (3 to 10% 02).Hydrogen is not required for microaerophilic growth. Growth occurs in the presence of 1 and 2% NaCl and 1% (wt/vol) pteridine vibriostatic compound 0/129. No growth occurs in the presence of 1% glycine and 0.1% 2,3,5-triphenyltetrazolium chloride. The strains have oxidase and catalase activities and reduce nitrate. Negative reactions in methyl red and VogesProskauer tests. Indole is not produced. Carbohydrates are neither fermented nor oxidized. Organic and amino acids are utilized as carbon sources. Hippurate, esculin, starch, and DNA are not hydrolyzed; gelatin is not liquified. Nonhemolytic. Menaquinone 6 and a second atypical menaquinone 6, the identity of which remains to be established, are the major respiratory quinones. Strains have been isolated from root-associated sediments and roots of salt march plants, from aborted fetuses of several species of farm animals, and from various other animal and human sources. Pathogenicity is unknown. The type species is Arcobacter nitrofigilis comb. nov. The DNA base composition ranges from 28 to 31 mol% G+C. Description of Arcobacter nitrofigilis comb. nov. Arcobacter nitrofigilis (basonym, Campylobacter nitrofigilis McClung, Patriquin, and Davis 1983) (ni. tro. fig’i. lis. L. n. nitrum, nitrate; L. v. figo, to fix; L. adj. suff.-ilis,able to; M. L. adj. nitrofigilis, able to fix [nitrogen] as nitrate). The description is the same as that given previously for Campylobacter nitrofigilis (32). Description of Arcobacter cryaerophilus comb. nov. Arcobacter cryaerophilus (basonym, Campylobacter cryaerophila Neill, Campbell, O’Brien, Weatherup, and Ellis 1985) (cry. ae. ro’ phi.lus. Gr. n. cruos, cold; Gr. n. aer, air; Gr. n. philos, friend; M. L. adj. cryaerophilus, friend of cold and air). The description is the same as that given previously for Campylobacter cryaerophila (37). rRNA cluster 111. rRNA cluster I11 contains members of four different genera (Helicobacter, Wolinella, “Flexispira,” and misnamed Campylobacter species) and strain CLO-3 of Fennell et al. (16). Wolinella succinogenes has a separate dendrogram (Fig. 1). The other taxa position on the TmCe) belonging to this rRNA branch have AT,,,, values of 6 to 10°C versus Wolinella succinogenes rRNA (Table 2). Furthermore, isoprenoid quinone composition (36) and several other characteristics (20) separate Wolinella succinogenes from the other taxa of this rRNA homology group. Strain CLO-3 is the only organism with a DNA base ratio similar to that of Wolinella succinogenes (Table 2); however, this is not necessarily an indication of a close relationship (9). Goodwin et al. (20) transferred [Campylobacter] pylori and [Campylobacter]mustelae to the new genus Helicobacter. The DNA-rRNA hydbridization results (Table 2 and Fig. 1) indicate that Helicobacter pylori, Helicobacter mustelae, [Campylobacter] cinaedi, and [Campylobacter]fennelliae are related to each other, at least at a level similar to the level between Campylobacter fetus and Campylobacter jejuni in rRNA cluster I (Fig. 1). Furthermore, all four species have G + C values between 35 and 41 mol% (Table 2) (20) and have many phenotypic characteristics in common (16, 20, 23, 59). Immunotyping (Table 4) and partial 16s rRNA sequence analysis (58) revealed that [Campylobacter] cinaedi and [Campylobacter]fennelliae are more closely related to each other than to Helicobacter pylori or Helicobacter mustelae. The number of flagella and fatty acid composition ([Cam-

INT. J . SYST.BACTERIOL.

pylobacter] cinaedi and [Campylobacter] fennelliae have tetradecanoic acid, hexadecanoic acid, and octadecenoic acid as major fatty acids, while Helicobacter pylori and Helicobacter mustelae have an additional 19-carbon cyclopropane fatty acid as a major fatty acid component) also differentiate [Campylobacter] cinaedi and [Campylobacter] fennelliae from Helicobacter pylori and Helicobacter mustelae. However, we believe that the genotypic and phenotypic similarities of the two Helicobacter species, [Campylobacter] cinaedi, and [Campylobacter]fennelliae outweigh their differences and justify inclusion of [Campylobacter] cinaedi and [Campylobacter]fennelliae in an emended genus Helicobacter. Emended description of the genus Helicobacter Goodwin, Armstrong, Chilvers, Peters, Collins, Sly, McConnell, and Harper 1989. Helical, curved, or straight unbranched gramnegative cells that are 0.3 to 1.0 pm wide and 1.5 to 5 pm long and have rounded ends and spiral periodicity. Nonsporeforming. Cells in old cultures may form spherical or coccoid bodies. Darting motility by means of a single polar flagellum (Helicobacter cinaedi and Helicobacter fennelliae) or multiple unipolar or bipolar and lateral flagella (Helicobacter pylori and Helicobacter mustelae). Flagella are sheathed (20, 23). Microaerophilic with a respiratory type of metabolism. Chemoorganotrophs. Carbohydrates are neither oxidized nor fermented. Energy is obtained from amino acids or tricarboxylic acid cycle intermediates but not from carbohydrates. Optimal growth occurs at 37°C in a humid atmosphere; no growth occurs at 25°C. Hydrogen is required or stimulates growth. No growth occurs in the presence of 3.5% NaCl. Growth occurs in the presence of 0.5% glycine and 0.04% triphenyltetrazolium chloride. Catalase and oxidase activities are present. No pigment production. No H,S production in triple sugar iron agar. No hydrolysis of hippurate. Susceptible to ampicillin, gentamicin, rifampin, and tetracycline; resistant to trimethoprim. Variable resistance to nalidixic acid and cephalothin (18, 20). The G + C content of the DNA ranges from 35 to 44 mol%. Isolated from gastric mucosa of humans and animals, from blood and feces of homosexual males, and from intestines of hamsters. Some organisms in this genus may be associated with gastritis and peptic ulcers in humans. The type species is Helicobacter pylori. Description of new Helicobacter species. The descriptions of Helicobacter cinaedi comb. nov. (basonym, Campylobacter cinaedi Totten, Fennell, Tenover, Wezenberg, Perine, Stamm, and Holmes 1985) and Helicobacter fennelliae comb. nov. (basonym, Campylobacter fennelliae Totten, Fennell, Tenover, Wezenberg, Perine, Stamm, and Holmes 1985) are the descriptions given by Totten et al. (59) for [Campylobacter] cinaedi and [Campylobacter] fennelliae, respectively. Taxonomic position of ‘‘Flexispira rappini” and strain CLO-3. “Flexispira rappini” and strain CLO-3 also belong to rRNA cluster I11 (Fig. 1).No significant precipitation values were found in the immunotyping analysis versus antisera of other taxa belonging to rRNA cluster I11 (Table 4). “Flexispira rappini” has sheathed flagella and a DNA base composition similar to the base compositions of Helicobacter strains (33 to 35 mol%) (Table 2). However, its unusual ultrastructure (spiral grooves) and several other phenotypic traits clearly distinguish this organism from the other taxa belonging to rRNA cluster 111(20). Strain CLO-3 has a DNA base ratio similar to that of Wolinella succinogenes (Table 2), but a ATm(e)of 9°C versus the latter organism (Fig. 1 and Table 2). Strain CLO-3 is also phenotypically different from

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TAXONOMY O F rRNA SUPERFAMILY VI

the other organisms of rRNA cluster I11 (20, 59). The relationships of strain CLO-3 and “Flexispira rappini” to the other taxa of rRNA cluster I11 remain to be investigated. Free-living Campylobacter-like strains. The saprophytic campylobacters of Laanbroek et al. (27) and Wolfe and Pfennig (65) do not belong to one of the three major rRNA homology groups and thus cannot be included within one of the five genera described above (Table 2 and Fig. 1). The DNA base ratios of [Spirillurn] sp. strain 5175 and Laanbroek strain CCUG 13942 are 38.4 and 41.6 mol%, respectively (27, 65). Both organisms are sulfur-reducing bacteria with a similar type of metabolism and are believed to belong to a group of saprophytic Campylobacter-like organisms which are isolated frequently from sediment samples of dirty freshwater or brackish water (41). Like all other members of rRNA superfamily VI, [Spirillurn] sp. strain 5175 has menaquinone 6 as one of its major respiratory quinones (7). As strain 5175 and strain CCUG 13942 are genotypically and phenotypically similar (41) and have similar taxonomic positions within rRNA superfamily VI (Fig. l),it does not seem unlikely that they belong to a single, separate genus within rRNA superfamily VI. ACKNOWLEDGMENTS J.D.L. is indebted to the Fonds voor Geneeskundig Wetenschappelijk Onderzoek, Belgium, for research and personnel grants. P.V. is indebted to the Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw, Belgium, for a scholarship and to the Nationaal Fonds voor Wetenschappelijk Onderzoek for a position as research assistant. We are indebted to H. Goossens and L. Vlaes for performing phenotypic tests on several CLO strains. E. F. is indebted to Lars Nehls, Ann Borjesson, and Marie Blomqvist for excellent technical assistance. REFERENCES 1. Archer, J. R., S. Romero, A. E. Ritchie, M. E. Hamacher, B. M. Steiner, J. H. Bryner, and R. F. Schell. 1988. Characterization of an unclassified microaerophilic bacterium associated with gastroenteritis. J. Clin. Microbiol. 26:lOl-105. 2. Beji, A., F. Mkgraud, P. Vincent, F. Gavini, D. Izard, and H. Leclerc. 1988. GC content of DNA of Carnpylobacter pylori and other species belonging or related to the genus Campylobacter. Ann. Inst. Pasteur/Microbiol. (Paris) 139527-534. 3. Bolton, F. J., D. Coates, D. N Hutchinson, and A. F. Godfree. 1987. A study of thermophilic campylobacters in a river system. J. Appl. Bacteriol. 62:167-176. 4. Bolton, F. J., A. V. Holt, and D. N. Hutchinson. 1985. Urease positive thermophilic campylobacters. Lancet i:1217-1218. 5. Bryner, J. H., A. E. Ritchie, L. Pollet, C. A. Kirkbride, and J. E. Collins. 1987. Experimental infection and abortion of pregnant guinea pigs with a unique Spirillum-like bacterium isolated from aborted ovine fetuses. Am. J. Vet. Res. 48:91-95. 6. Collins, M. D., and D. Jones. 1981. Distribution of isoprenoid quinone structural types in bacteria and their taxonomic implications. Microbiol. Rev. 45316-354. 7. Collins, M. D., and F. Widdel. 1986. Respiratory quinones of sulphate-reducing and sulphur reducing bacteria: a systematic investigation. Syst. Appl. Microbiol. 8:8-18. 8. De Ley, J. 1970. Reexamination of the association between melting point, buoyant density, and chemical base composition of deoxyribonucleic acid. J. Bacteriol. 101:738-754. 9. De Ley, J. 1978. Modern molecular methods in bacterial taxonomy: evaluation, application, prospects, p. 347-357. In Proceedings of the 4th International Conference of Plant Pathogenic Bacteria, Angers, vol. 1. Gibert-Clarey, Tours, France. 10. De Ley, J., H. Cattoir, and A. Reynaerts. 1970. The quantitative

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Revision of Campylobacter, Helicobacter, and Wolinella taxonomy: emendation of generic descriptions and proposal of Arcobacter gen. nov.

Hybridization experiments were carried out between DNAs from more than 70 strains of Campylobacter spp. and related taxa and either 3H-labeled 23S rRN...
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