INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1988, p. 190-200 Vol. 38. No. 2 0020-77 13/88/020190-11$02.OO/O Copyright 0 1988, International Union of Microbiological Societies

Phylogenetic Study of the Genus LOUIS M. THOMPSON 111,’ ROBERT M. SMIBERT,2 JOHN L. JOHNSON,2 AND NOEL R. KRIEG1* Microbiology and Immunology Section, Department of Biology,’ and Department of Anaerobic Microbiology,2 Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061

The phylogenetic relationships of all species in the genus Cantpylobacter, Wolinella succinogenes, and other gram-negative were determined by comparison of partial 16s ribosomal ribonucleic acid sequences. The results of this study indicate that species now recognized in the genus Campylobacter make up three separate ribosomal ribonucleic acid sequence homology groups. Homology group I contains the following true Campylobacter species: Campylobacterfetus (type species), Campylobacter coli, Campylobacter jejuni, Campylo- bacter laridis, Campylobacter hyointestinalis, , , Campylobacter sputorum, and ‘‘Campylobacter upsaliensis” (CNW strains). “Campylobacter cinaedi,” ‘Campylobacter fennelliue,” Campylobacter pylori, and W.succinogenes constitute homology group 11. Homology group I11 contains Campylobacter ctyaerophiza and Campylobacter nitrofigilis. We consider the three homology groups to represent separate genera. However, at present, easily determinable phenotypic characteristics needed to clearly differentiate them are not apparent. The three homology groups are only distantly related to representatives of the alpha, beta, and gamma branches of the purple bacteria, indicating that these bacteria do not belong to any previously defined branch of this phylum.

Campylobacters have been recognized for many years as the CNW strains represent a distinct species (36). Campylo- important agents of reproductive diseases in sheep and bacter cryaerophila (sic) is an aerobic species that causes cattle, and some of the species have also been found to cause abortions in pigs, cattle, horses, and sheep and can occa- diseases in humans. The type species of the genus, Cam- sionally be isolated from human infections (17, 27), whereas pylobacter fetus, is divided into two subspecies; C. fetus Campylobacter nitrofigilis is a microaerophilic, NaC1-re- subsp. venerealis causes sexually transmitted abortions in quiring, nitrogen-fixing bacterium found in the roots of salt cattle, and C. fetus subsp. fetus causes orally transmitted marsh grasses (25). sporadic abortions in cattle and sheep and blood infections in The classification of the genus Campylobacter has always humans (39). Campylobacter hyointestinalis has been iso- been somewhat difficult. Because these organisms do not lated from pigs with proliferative ileitis (8) and occasionally catabolize carbohydrates and are inert with regard to most from homosexual males with proctitis (5). Campylobacter traditional biochemical tests used for the identification of mucosalis has been isolated from lesions of porcine intesti- bacteria, only a relatively small number of tests are available nal adenomatosis (8, 20, 39). Campylobacter jejuni is part of for the identification and classification of the normal intestinal flora of cattle, sheep, dogs, cats, (35, 36). Consequently, classification of campylobacters poultry, and other animals (39), and it is a major cause of based on only a few biochemical and physiological tests bacterial gastroenteritis in humans (3). Campylobacter coli is gives few means to accurately differentiate species. part of the normal flora of pigs and poultry and can also Although DNA homology studies have shown that the Campylobacter laridis cause diarrhea in humans. occurs in current Campylobacter species are distinct from one another the intestines of sea gulls, humans, dogs, and horses and (1, 2, 9, 11, 21, 30, 31, 35, 36, 47), they have not answered occasionally causes blood infections and diarrhea in humans the question of whether these species are sufficiently related (15, 26, 38, 45). Campylobacter concisus has been isolated from the oral cavities of people with periodontal disease (42). to justify classification within a single genus. Comparison of Campylobacter sputorum, which is divided into three bio- 16s ribosomal ribonucleic acid (rRNA) sequences for phy- vars, biovars sputorum, bubulus, and fecalis, occurs as part logenetic analysis has proven to be a powerful tool for of the normal flora of human mouths, bovine genitalia, and accurate classification of microorganisms above the level of sheep feces, respectively (36). Campylobacter pylori is a species (7, 29, 40, 41, 49, 50). The recent development of a probable causative agent of gastric and duodenal ulcers and technique which facilitates the rapid generation of partial chronic gastritis in humans (22, 23). “Campylobacter 16s rRNA sequences has allowed researchers to determine cinaedi” and “Campylobacter fennelliae” have been asso- the phylogenetic relationships among bacteria (18). Using ciated with proctitis, proctocolitis, enteritis, and bacteremia this technique, Romaniuk et al. (34) concluded that C.pylori in homosexual men (4,6,28,46).CNW (catalase-negative or is not related at the genus level to C.jejuni, C. coli, C.fetus weakly catalase-positive) strains of Campylobacter have subsp.fetus, C. laridis, or C. sputorum biovar sputorum and been isolated from healthy and diarrheic dogs and cats (37; that these latter species represent the true genus Campylo- C. J. Gebhart, G. E. Ward, and L. A. Finsmith, Abstr. bacter. C. pylori was more closely related to Wolinella Annu. Meet. Am. SOC. Microbiol. 1984, C55, p. 245); the succinogenes than to the other campylobacters. Using data name “Campylobacter upsaliensis” has been suggested for based on partial 16s rRNA sequences, Lau et al. (19) these organisms (K. Sandstedt and J. Ursing, XIV Int. reported that C. jejuni, C. coli, and C. laridis are very Congr. Microbiol., 1986, abstr. no. P.B8-17). Deoxyribonu- closely related, that the C. jejuni-C. coli-C. laridis group, C. cleic acid (DNA) reassociation experiments have shown that fetus, C. sputorum biovar fecalis, and C. pylori are all distinct from one another, and that W. succinogenes is related to the campylobacters. More recently, Paster and * Corresponding author. Dewhirst (32) confirmed the relationship between W. succi-

190 VOL. 38, 1988 PHYLOGENETIC STUDY OF CAMPYLOBACTER 191

TABLE 1. Strains of Cutnp~lohucrerspecies used in this study isolated by a modification of a previously described proce- Species“ Strain“ dure (14. 16). Lysates of cells disrupted with a French pressure cell were extracted with a phenol-cresol solution; Catalase-positive campylobacters 16s rRNA was selectively precipitated from the soluble C. coli ...... ATCC 33559’ 7.5 C. jejirtti ...... ATCC 33560’ ribonucleic acid (RNA) and DNA by adding cold (-20°C) C. ferrrs subsp. .fitus ...... ATCC 27374’ M ammonium acetate. The rRNA was stored at -20°C in a C. .fetzr.s subsp. venereulis ...... ATCC 19438’ buffer consisting of 0.15 M NaCI, 0.01 M sodium ethylene- C. hyointestiticilis ...... 80-4577-4 (= ATCC diaminetetraacetate, 1.0 mM N-2-hydroxyethylpiperazine- 35217Ih N’-2-ethanesulfonic acid (HEPES buffer), and 1.0% sodium C. luridis ...... NCTC 11352T dodecyl sulfate. The sodium dodecyl sulfate was included to C. cryueropttila ...... NCTC lltWT inhibit the activity of ribonucleases; its omission resulted in C. nitrojgilis...... ATCC 33309T RNA degradation even at -20°C. “C. cinaedi” ...... ATCC 35683‘ Synthesis and purification of oligonucleotide primers. Prim- “C. fennelliue” ...... ATCC 35684‘ C. py/ori ...... NCTC 11637T ers complementary to conserved regions of the 16s rRNA Cataiase-negative campylobacters molecule were prepared by using a DNA synthesizer (model C. mrrcoscilis ...... NCTC 11000’ 381A: Applied Biosystems, Foster City, Calif.). The five C. conc.isu.s...... VPI 13086T primers used in this study were complementary to the C. sputoriim biovar sputorum ...... VPI S-17 (= ATCC following regions of Eschericlzin cdi 16s rRNA (5’ to 3’): 35980)“ positions 321 to 340,519 to 536,907 to 926,1220 to 1239, and CNW (“C. rrpsaliensis”) ...... CG-1” 1388 to 1407. Crude oligonucleotide preparations were puri- ‘‘ ATCC. American Type Culture Collection, Rockville. Md.: NCTC. fied by thin-layer chromatography (Brian Reid, personal National Collection of Type Cultures. London. England: VPI. Virginia communication). diluted to a concentration of 0.1 mg/ml Polytechnic Institute and State University. Blacksburg: CNW. catalase neg- with TE buffer [lo mM tris(hydroxyrnethy1)aminomethane ative or weakly positive: T = type strain. base and 0.1 mM ethylenediaminetetraacetate, pH 8.01, and ” Type strain proposed by Gebhart et al. (8). ‘ Type strain proposed by Totten et al. (46). stored at -20°C. ‘‘ Neotype strain proposed by Roop et al. (36). Preparation of RNA for sequencing. The sodium dodecyl ‘’ Reference strain C. J. Gebhart. G. E. Ward, and L. A. Finsmith. Abstr. sulfate present in the rRNA storage buffer was removed Annu. Meet. Am. Soc. Microbiol. 1984, CSS, p. 245). prior to base sequencing by extraction with a phenol-chlo- roform-salts solution. This was followed by extraction with nogenes and C. pylori based on analysis of partial 16s rRNA secondary butanol to remove any residual phenol. The sequences and also reported that Wolinelln recto. Wolinellcr rRNA was precipitated in 2 volumes of cold (-20°C) 95% ciirvn , Bacteroides gracilis, and Bncteroides iireolyticiis ethanol and dissolved in TE buffer to a concentration of 2.0 belong to the same cluster as the true campylobacters. mg of RNA per ml. The partial 16s rRNA sequence analyses reported to date Determination of RNA base sequences. Partial 16s rRNA are incomplete because they have included only a limited sequences were determined by a procedure based on that of number of Campylobncter species. We report here the Lane et al. (18). Certain modifications in this procedure (see results of comparisons of partial 16s rRNA sequences from below) were made to enhance the resolution of base se- all 14 species of Cnmpylohac*ter and the relationships of quence ambiguities. Microfuge tubes (0.5 ml) served as these organisms to one another and to other gram-negative reaction vessels throughout the procedure. Oligonucleotide bacteria. primers were hybridized to the 16s rRNA template in a reaction mixture consisting of 1.0 pl of RNA (2.0 pg/pl), 2.0 pl pI 5x MATERIALS AND METHODS of primer (0.1 pg/pl), 1.0 of hybridization buffer [250 mM tris(hydroxymethy1)aminomethane hydrochloride, Bacterial strains and growth of stock cultures. The Cmm- 500 mM KCI, pH 8.51. and 1.0 pl of water. The reaction pylobacfer strains used in this study are listed in Table 1. mixtures were heated at 90°C for 2 min, allowed to cool to With the exception of the CNW group, the type or proposed 60°C over a period of 30 min, and then placed on ice. To this neotype strain of each species was used. The identity of each mixture we added 5.0 pl of 5~ reverse transcriptase buffer strain was confirmed by differential biochemical tests (35, [250 mM tris(hydroxymethy1)aminomethane hydrochloride, 36) or by comparison of its rRNA sequence with previously 250 mM KCl, 50 mM dithiothreitol, 50 mM MgCl,, pH 8.31, published sequences (34). C. coli, C. jejirni, C. fetiis subsp. 2.0 p,l of [35S]deoxyadenosine triphosphate ([3sS]dATP; 10 fetus, C. jetus subsp. venererrlis, C. liyointestinulis, C. pCi/pI). 1.0 pl of avian myeloblastosis virus reverse tran- Inridis, C. spirtorurn biovar sputorum, and CNW strain CG-1 scriptase (9 U/pI; Promega Biotec), and 2.0 pl of water. A were cultured under an air atmosphere at 37°C in brucella 3-p1 portion of this mixture was added to each of four semisolid medium (brucella broth [GIBCO Laboratories, reaction vessels containing 2.0 pl of a solution consisting of Grand Island, N .Y.] containing 0.15% agar). C. c*iynerophila 10 pM dATP, 250 pM deoxycytidine triphosphate, 250 pM and C. nitrojigilis were cultured in brucella semisolid me- deoxyguanosine triphosphate, 250 pM deoxythymidine tri- dium under an air atmosphere at 30°C. The medium for C. phosphate, and one of the following dideoxynucleoside nitrojigilis was supplemented with 1.0% NaCI. C. nzircosalis, triphosphates: 1.25 pM dideoxyadenosine triphosphate, 5.0 C. concisus, C. pylori, “C. cinnedi.” and “C. fennelliae” pM dideoxycytidine triphosphate, 7.5 pM dideoxyguanosine were cultured in brucella semisolid medium at 37°C under an triphosphate, or 10 pM dideoxythymidine triphosphate. The atmosphere containing 6% 02,10% CO?, 15% HZ,and 69% reaction mixtures were incubated at 48°C for 20 min. A 1-p.1 N,. The medium for C. nirrcosnlis and C. concisirs was portion of a chase mixture [1.0 mM concentration of each supplemented with 0.3% fumaric acid. deoxynucleotide triphosphate, 0.2 U of avian myeloblastosis Isolation of RNA. Biphasic culture systems employing virus reverse transcriptase per pl, 10 mM tris(hydroxy- Roux bottles were used for the growth of all strains, as methy1)aminomethane hydrochloride, pH 8.31 was added, described by Roop et al. (35, 36). Bulk cellular rRNA was and the mixtures were incubated for an additional 15 min. 192 THOMPSON ET AL. INT. J. SYST.BACTERIOL.

Reactions were stopped by adding 6.0 pl of stop mixture homology values of 292.0%. Subgroup 2 includes C. fetus, (86% formamide, 10 mM ethylenediaminetetraacetate,0.08% C. hyointestinalis, C. concisus, C. mucosalis, and C. sputo- xylene cyanol, 0.08% bromophenol blue). Complementary rum, which have interspecies homology values of 288.0%. DNA was denatured from the RNA template by heating each Group I1 contains “C. cinaedi,” “C. fennelliae,” and C. sample at 90°C for 2 min immediately prior to loading the pylori, which have interspecies homology values ranging sample on the sequencing gel. The sequencing gels were 8% from 82.5 to 88.8%. Group I1 also includes W. succinogenes, polyacrylamide-8 M urea buffer gradient gels and were 0.4 which exhibits levels of homology of 80.1 to 86.8% with the mm thick and 40 cm long. three Campylobacter species. To improve the analysis for campylobacters, certain as- Group I11 contains C. cryaerophila and C. nitrofigilis, pects of the procedure described above, as determined by which have an interspecies homology value of 86.9%. experimentation, differed from the procedure described by The homology level between groups I and I1 is 72.4%. Lane et al. (18). (i) Optimal incorporation of [35S]dATPinto Groups I and I1 are related to group I11 at a level of 68.8%. the complementary DNA occurred when 20 pCi per sample The levels of partial 16s rRNA sequence homology among was used instead of 30 pCi per sample. Also, the concentra- the three major branches of the purple phototrophic bacteria tion of dATP was decreased from 125 to 10 pM, and this, were determined by comparing the known sequences for together with the decreased level of [35S]dATP, greatly representatives of each branch. A. tumefaciens and P. improved the extension of the complementary DNA tran- testosteroni were related at a level of 56.0%, A. tumefaciens script while still allowing sufficient incorporation of the and E. coli were related at a level of 58.2%, and E. coli and [35S]dATP.(ii) The temperature of the reverse transcriptase P. testosteroni were related at a level of 57.0%. Groups I, 11, reaction was increased from 37 to 48°C to minimize the band and I11 exhibited a level of rRNA sequence homology of anomalies (presumably attributable to the secondary struc- 49.3% with the above three representative species. ture of the rRNA) that occurred across all four lanes of the sequencing gel when the lower temperature was used. Tem- DISCUSSION peratures higher than 48°C were less effective, probably because of degradation of the reverse transcriptase. (iii) To In general, our results agree with those of Romaniuk et al. minimize nonspecific binding of the primers to the template (34), Lau et al. (19), and Paster and Dewhirst (32). In the rRNA, the samples were heated at 90°C for 2 min instead of present study, all species of the genus Campylobacter were 1 min, and they were allowed to cool to 60°C over a 30-min included, and all were represented by type strains (or, in the period instead of to 25°C over a 10-min period. case of the CNW group, an established reference strain). Comparison of RNA sequences. Sequence homologies were Lau et al. (19) did not use type strains in their study. In calculated by using methods described previously (24, 29). investigations of this type it is essential that type strains, Positions of unknown base composition and ambiguous which are the permanent examples of the species, be used regions of alignment were eliminated prior to homology rather than clinical isolates that may not be identified cor- calculations. Alignment gaps were assigned one-half the rectly. The low intergroup rRNA sequence homology values value of a nucleotide base in the same position. Similarity among groups I, 11, and I11 indicate that these groups should coefficients were calculated, and a dendrogram was con- not continue to be included within a single genus. Because structed by using the Numerical System (NT- group I contains the type species, C. fetus, only this group SYS) analysis program (33). could represent the genus Campylobacter. The finding that one subgroup within group I contains C. RESULTS coli, C. jejuni, and C. laridis is consistent with previous data from DNA reassociation experiments; these three species The partial 16s rRNA sequences determined for the 14 were more closely related to one another as determined by Campylobucter species are aligned in Fig. 1 with the known DNA reassociation than to any other Campylobacter species sequences for representatives of the three major branches of (1, 31, 35, 47). Our findings indicate that CNW strain CG-1, the purple bacteria-Agrobacterium tumefaciens (alpha which represents a distinct species as determined by DNA branch), Pseudomonas testosteroni (beta branch), and E. homology data (36), also belongs to this subgroup. A com- coli (gamma branch) (52)-and W. succinogenes (19). The parison of the phenotypic properties of the four species levels of similarity of the aligned sequences after removal of indicates that they can all grow at 42°C (2, 10, 35, 36, 39) and all base positions of unknown composition and all ambigu- that they are all catalase positive except the CNW strains, ous sequence alignments are shown in the upper portion of which may give a negative, weakly positive, or delayed Table 2. The 14 Campylobacter species could be assigned to reaction depending on the cultural conditions (37). three distinct rRNA sequence homology groups. The three The second subgroup within group 1 includes C. fetus and groups are especially apparent when the positions of base C. hyointestinalis; this is consistent with previous data from sequences common to all of the organisms are removed from DNA reassociation experiments which indicated that these consideration (lower portion of Table 2). Such common base two species are related (36). Our findings indicate that C. sequences are of little phylogenetic value (48). Organisms concisus, C. mucosalis, and C. sputorum also belong to this that are closely related continue to exhibit high levels of subgroup. A phenotypic comparison indicates that all of the rRNA sequence homology. The relationships among the species except C. fetus can grow at 42”C, all except C. fetus various species are represented graphically in Fig. 2. subsp. venerealis can grow in the presence of 1% glycine, Group I contains the following nine species: C. coli, C. and all except C. concisus are susceptible to cephalothin (35, jejuni, C. laridis, CNW strain CG-1, C.fetus (both subspe- 36). cies), C. hyointestinalis, C. concisus, C. mucosalis, and C. Group I1 contains C. pylori, “C. cinaedi,” “C. fennel- sputorurn. These species have interspecies homology values he,” and W. succinogenes. Our results confirm those of ranging from 83.2 to 96.8%. Group I could be further divided Romaniuk et al. (34), Lau et al. (19), and Paster and into two subgroups. Subgroup 1 includes C. coli, C.jejuni, Dewhirst (32), who previously indicated that C. pylori C. laridis, and CNW strain CG-1, which have interspecies should be excluded from the genus Campylobacter. Our VOL.38, 1988 PHYLOGENETIC STUDY OF CAMPYLOBACTER 193

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TABLE 2. Homology values basedon partial 16s rRNA sequences of Campylobacterspecies and other procaryoticspecies % Homologyto:*

C. A. Taxon c. c. c. :ugr C. hyoin- C. con- C. muco- C.sputa- CNW C. C.fen- C. W.succi- nitroji- E. coli ’* teroni turn&- coli jejuni laridis fetus venere- testinalis cisus salis rum CG-1 cinaedi nelliae pylori nogenes gilis ciens alis

~~~~ ~ ____~ ~ ~ ~~~ C. coli 98.1 96.8 96.5 96.4 97.7 96.4 95.7 94.7 96.7 90.6 88.1 87.4 88.7 87.4 88.2 78.5 78.1 78.1 C.jejuni 96.8 97.7 96.0 95.8 97.3 95.8 95.1 94.8 97.5 90.0 87.6 86.7 88.5 88.1 88.8 79.2 79.7 79.0 C. laridis 93.6 96.0 94.5 94.6 95.8 95.0 93.4 93.2 96.6 89.0 86.8 85.2 87.5 86.5 87.3 77.6 78.5 78.4 C.fetus subsp. 92.4 90.4 88.0 98.5 98.0 98.0 97.7 95.5 94.2 90.4 89.0 86.6 88.9 87.7 87.7 79.4 78.9 78.1 fetus C.fetus subsp. 92.0 90.0 88.4 96.4 97.9 97.9 97.7 95.5 94.4 90.3 88.7 86.3 88.9 87.9 87.5 79.4 79.1 78.1 venerealis C. hyointestinalis 95.2 93.6 91.6 95.2 94.8 98.0 97.2 95.5 95.9 90.4 88.9 86.8 88.5 87.9 87.9 79.0 78.3 78.5 C. concisus 90.8 89.6 87.6 94.4 94.0 94.0 97.4 96.2 93.9 90.8 89.7 87.4 89.2 88.2 88.4 79.9 79.8 79.1 C. mucosalis 89.6 87.6 84.4 93.6 92.8 92.4 92.8 94.9 92.9 89.4 87.9 86.4 87.9 87.7 87.1 79.4 78.1 77.6 C. sputorum 88.4 87.6 85.6 90.0 89.6 89.6 89.2 88.0 92.6 89.6 88.2 87.3 88.7 87.1 87.5 78.8 78.6 77.0 CNW strain CG-1 92.4 94.0 92.0 86.4 86.0 88.8 86.0 83.2 83.6 89.0 87.4 86.7 87.2 86.8 87.2 78.5 79.0 79.0 “C. cinaedi” 77.5 76.1 74.5 78.0 77.2 77.6 77.6 74.8 75.9 74.9 95.2 92.5 92.4 84.8 85.4 79.2 78.7 79.2 “C.fennelliae” 72.1 70.1 69.3 73.7 73.3 73.3 74.1 71.7 72.1 71.3 88.8 93.4 94.2 84.1 85.7 79.4 78.7 79.6 C. pylori 69.0 69.1 67.1 69.0 68.2 69.8 71.0 68.6 69.8 68.3 82.5 85.7 91.5 83.7 83.7 77.8 77.6 78.3 W.succinogenes 73.5 72.4 70.0 72.8 72.3 72.3 74.4 71.5 72.8 70.0 82.4 86.8 80.1 86.2 87.2 79.4 79.1 80.1 C. cryaerophila 69.5 70.9 68.4 71.3 72.1 71.3 72.5 71.3 69.7 68.5 63.7 62.2 62.3 67.6 94.2 78.6 77.8 78.3 C. nitrojigilis 71.3 72.1 69.7 70.9 71.7 70.5 72.1 69.3 69.7 69.7 65.3 63.8 61.1 68.8 86.9 79.5 78.0 78.5 E. coli 48.6 49.5 47.9 50.6 50.2 49.8 52.2 49.8 49.0 49.0 50.6 49.9 47.3 51.7 49.5 51.5 81.9 82.4 P. testosteroni 48.0 50.5 49.7 49.2 49.2 49.2 51.6 48.0 48.8 51.7 50.8 49.3 47.9 50.7 47.3 46.9 57.0 81.4 A. tumefaciens 48.0 49.3 50.1 47.6 46.8 48.0 50.0 47.2 45.2 50.8 49.6 50.1 48.9 52.3 49.1 48.9 58.2 56.0

~ ~~ a The values in the upper portionof the table are thelevels of similarityof the alignedsequences after removalof all base positionsof unknowncomposition and all ambiguous sequence alignments, whichleft approximately750 nucleotide basesper organism forcomparison. Alignment gaps wereassigned one-half the valueof a nucleotidein the same position.The valuesin the lower portionof the tablewere determinedin a similarfashion, except that all positionsof constant base composition werealso eliminated from consideration, whichleft approximately250 nucleotide basesper organism forcomparison.

CL W4 198 THOMPSON ET AL. INT. J. SYST.BACTERIOL.

c. coli C. jejuni C. laridis CNW CG-I C. fetus fetus C. fetus venerealis C. hyointestinalis C. concisus C. mucosalis C. sputorum C. cinoedi C. fennelliae w succinogenes C. PYlWi C. cryaerop hila r b. nitrofigilis E. coli A. tumefaciens P. testosteroni

40.3 55.3 623 69.3 76.3 033 90.3 97.3

PERCENT HOMOLOGY FIG. 2. Phylogenetic relationships of Carnpyfobacter species, W. succinogenes, and representatives of the major branches of the purple phototrophic bacteria, based on analysis of partial base sequences of 16s rRNA. The horizontal scale is the percent homology calculated by the Numerical Taxonomy System (NTSYS) analysis program, using the values listed in the lower portion of Table 2. results further indicate that the level of rRNA sequence and are not true wolinellas; instead, they are closely related homology between C. pylori and W. succinogenes is 80.1% to C. jejuni, C. coli, C. laridis, C.fetus, C. concisus, and C. and that, as a group, W. succinogenes, “C. ciulaedi,” and spurorurn. Thus, they belong to homology group I. More- “C. fennelliae” are related to C. pylori at a level of 82.8%. over, Paster and Dewhirst indicated that two other putative Because the error in the RNA sequencing procedure used in anaerobes, B. gracilis and B. ureolyticus, also are closely this study is approximately 1.0% (18), it seems arbitrary to related to the true campylobacters. Consequently, these exclude any of these species from the genus represented by species should also be included in homology group I. group 11. W. succinogenes is the type species of the genus Whether all of these species are microaerophilic requires Wolinella, and thus the genus represented by group I1 should investigation. bear the name Wolinella. W. succinogenes is presently Group I11 consists of C. cryaerophila and C. tzitrojigilis, defined as being anaerobic, catalase negative, and H,S which are related at an rRNA sequence homology value of positive, with a guanine-plus-cytosine content of 42 to 48 86.9%. In view of the fact that C. cryaerophila is an aerobe mol% (44). However, in 1961, Wolin et al. (51) clearly that causes abortion in pigs and other animals and occasion- showed that W. succinogenes (then called “ Vibrio succino- ally causes blood infections in humans, whereas C. nitroji- genes”) is oxidase positive and is capable of using 0, as a gilis is a microaerophilic, NaC1-requiring nitrogen fixer as- terminal electron acceptor under microaerobic conditions sociated with the roots of marsh grasses, a phenotypic (approximately 2% O,), but not under atmospheric levels of definition of the genus represented by these two species is 0,. Regardless of whether fumarate may be a more efficient elusive. However, now that the relationship between the two terminal electron acceptor than 0,, the fact remains that species is known, further characterization studies might anaerobes are not capable of using 0, as a terminal electron reveal unifying phenotypic similarities. For instance, both acceptor for respiration. These findings, along with addi- species have optimal growth temperatures below 37”C, and tional evidence on the electron transport system (12, 13), they can grow at temperatures as low as 6°C (25, 27). indicate that W. succinogenes is not an anaerobe but is Moreover, it is interesting that 80% of the C. nitrojigilis instead an H,-requiring microaerophile. C. pylori, “C. strains characterized by McClung et al. (25) exhibited urease cinaedi, ” and “C. fennelliae” are microaerophilic, grow activity and that 75% of the strains grew in 1% bile. Such best in a microaerobic atmosphere containing H, (data not features might be indicative of a bacterium which has an shown), are catalase positive and H,S negative, and have a animal host, and this suggests that the habitat of this species guanine-plus-cytosine content of 37 to 38 mol%. Conse- might not be (or might not have been) limited to marsh grass quently, the description of the genus Wolinella should be roots. emended to include these species. The difficulty in arriving at suitable, mutually exclusive Results recently obtained by Paster and Dewhirst (32) phenotypic descriptions of the genera and some species indicate that the other species of WulineIla ( W. recta [42] and represented by groups I, 11, and I11 can be attributed to the W. curva [43]) are not closely related to W. succinogenes present paucity of phenotypic characteristics for campylo- VOL. 38, 1988 PHYLOGENETIC STUDY OF CAMPYLOBACTER 199 bacters and Wolinella and also to the fact that not all of the biochemical characteristics and a biotyping proposal for Cam- organisms have been compared by the same tests and pylobacter jejuni. J. Clin. Microbiol. 151065-1073. methods. Now that the relationships among the campylobac- 11. Hebert, G. A., D. G. Hollis, R. E. Weaver, A. G. Steigerwalt, ters have been delineated, it is to be hoped that unique R. M. McKinney, and D. J. Brenner. 1983. Serogroups of differential phenotypic features of each genus represented by Campylobacter jejuni, Campylobacter coli, and Campylobacter fetus defined by direct immunofluorescence. J. Clin. Microbiol. the three rRNA sequence homology groups can be discov- 17:529-538. ered. 12. Jacobs, N. J., and M. J. Wolin. 1963. Electron-transport system It is not yet clear just where campylobacters belong among of Vibrio succinogenes. I. Enzymes and cytochromes of the the eubacteria. The fact that groups I, 11, and I11 are related electron-transport system. Biochim. Biophys. Acta 69:18-28. to one another at a level of 68% strongly suggests that the 13. Jacobs, N. J., and M. J. Wolin. 1963. Electron-transport system three groups belong together on the same phylogenetic of Vibrio succinogenes. 11. Inhibition of electron transport by branch. Romaniuk et al. (34) indicated that campylobacters 2-heptyl-4-hydroxyquinoline N-oxide. Biochim. Biophys. Acta appear to belong to a previously undefined phylogenetic 69:29-39. 14. Johnson, J. L. 1981. Gsrietic characterization, p. 450472. In P. branch, based on a signature sequence analysis and on a Gerhardt, R. G. E. >Jui-ray, R. N. Costilow, E. W. Nester, comparison of sequence data with partial sequences from W. A. Wood, N. R. KIieg, and G. B. Phillips (ed.), Manual of representatives of 8 of the 10 major phylogenetic branches of methods for general bxteriology, American Society for Micro- the eubacteria. Our data indicate that groups I, 11, and I11 biology, Washington, L.i .C. have an overall rRNA sequence similarity of 49.3% to 15. Karmali, M. A., and P. C. Fleming. 1979. Campylobacter representatives of the alpha, beta, and gamma phylogenetic enteritis. Can. Med. Assoc. J. 120:1525-1532. branches of the purple bacteria. This is a depth of divergence 16. Kirby, K. S. 1965. 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