INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1991, p. 31-38 Vol. 41, No. 1 0020-7713/9 1/01003 1-08$02.00/0 Copyright 0 1991, International Union of Microbiological Societies

Phylogeny of felis sp. nov. , Helicobacter mustelae, and Related B. J. PASTER,l* A. LEE,2 J. G. FOX,3 F. E. DEWHIRST,l L. A. TORDOFF,l G. J. FRASER,l J. L. O’ROURKE,* N. S. TAYLOR,3 AND R. FERRER02 Forsyth Dental Center, Boston, Massachusetts 02115l; The University of New South Wales, Sydney, New South Wales, Australia 20332; and Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 021393

Strain CSIT (T = type strain) is a gram-negative, microaerophilic, -positive,spiral-shaped bacterium that was isolated from the gastric mucosa of a cat. Additional strains which possessed biochemical and ultrastructural characteristics similar to those of strain CSITwere isolated from the gastric mucosa of cats and dogs. The guanine-plus-cytosine content of the DNA of strain CSIT was 42.5 mol%. The 16s rRNA sequences of strain CSIT, strain DS3 (a spiral-shaped isolate from a dog), and Helicobacter musteke were determined by direct RNA sequencing, using a modified Sanger method. These sequences were compared with the 16s rRNA sequences of Helicobacter pylon’, “Flexispira rappini, ” Wolinella succinogenes, and 11 of campylobac- ters. A dendrogram was constructed based upon sequence similarities. Strains CSIT and DS3 were very closely related (level of similarity, 99.3%). Two major phylogenetic groups were formed; one group consisted of strains CSIT and DS3, H. mustelae, H. pylori, “F. rappini,” and W. succinogenes, and the other group contained the true . The average level of similarity between members of these two groups was 84.9%. Within the first group, strains CSIT and DS3, H. pylon’, and H. mustelae formed a cluster of organisms with an interspecies similarity level of 94.5% The phylogenetic positions of W. succinogenes and “F. rappini” were just outside this cluster. On the basis of the results of this study, we believe that strains CSIT (= ATCC 49179T) and DS3 represent a new species of the Helicobacter, for which we propose the name .

Cumpylobacter species commonly colonize the alimentary have their own distinctive gastric floras (11, 23). A gram- and genital tracts of and , and some are negative, microaerophilic, urease-positive, spiral-shaped rod responsible for gastrointestinal diseases (32). It has been (strain CSIT [T = type strain]) was isolated from the gastric proposed that the mucus lining the gut mucosa is the mucosa of a cat and has been described previously (28). ecological niche inhabited by these bacteria (22, 25). Re- Strain CSIT is physiologically similar to, but morphologi- cently, increased attention has been focused on the species cally different from, H. pylori and H. mustelae. The gastric isolated from the stomachs of mammals, including humans bacteria isolated thus far have different distinctive morphol- (14,24). The gastric mucus appears to be the natural habitat ogies with respect to cell shape and number and arrangement of , a bacterium associated with histolog- of flagella (11, 17). ical in human stomachs (27, 31), and Helicobacter In this study, six additional bacterial strains were isolated mustelae, an organism associated with gastritis and ulcers in from the gastric mucosa of dogs and cats. These isolates adult ferrets (9, 10). On the basis of 16s rRNA sequencing were similar to strain CSIT with respect to ultrastructural data and biochemical and phenotypic criteria, these two and biochemical characteristics. The partial 16s rRNA se- species were recently transferred from the genus Campylo- quences for the cat gastric spirillum (strain CSIT) and for one bacter to the genus Helicobacter (13). The overall phylogeny dog isolate (strain DS3) were determined in order to estab- of members of the genus and related bacteria lish their phylogenetic position among the campylobacters, has been studied extensively (21, 34, 36, 40). These bacteria H. pylori, and related bacteria. As discussed below, the fall into three phylogenetic groups. The first group comprises name Helicobacter felis is proposed for these spirillumlike the true campylobacters, including , organisms, and we refer to them below by the proposed subspecies of Campylobacter fetus, Campylobacter hy- name, In addition, we present the 16s rRNA sequence of H. ointestinalis, Campy lobacter concisus, Campylobacter mu- mustelae since the phylogenetic relationship of this organism cosalis, Campylobacter sputorum, Campylobacter coli, to other bacteria was uncertain previously. Campylobacter lari, Wolinella recta, Wolinella curva, and two misclassified bacteroides, Bacteroides gracilis and Bac- MATERIALS AND METHODS teroides ureoiyticus. The second group contains H. pylori, H. mustelae, Wolinella succinogenes, and two misclassified Bacterial strains and culture conditions. Strains of H. felis campylobacters, Campylobacter cinaedi and Campylobac- were grown in brain heart infusion broth (Difco Laborato- ter fennelliae (13, 40; unpublished data). A third group, ries, Detroit, Mich.) supplemented with 5% sterile fetal calf which presently consists of only two species, Campylobac- serum (Sigma Chemical Co., St. Louis, Mo.) and an antibi- ter cryaerophila and Campylobacter nitrofigilis, branches off otic combination consisting of vancomycin (10 ng/ml), poly- from the other two groups at a deeper phylogenetic level mycin (2.5 pg/ml), trimethoprim (5 pg/ml), and amphotericin (40). (2 kg/ml). Flasks containing liquid media were evacuated Many animal species, such as dogs, cats, and primates, once to a level of 26 in. (ca. 66 cm) of Hg and filled with a 5% co2-5% H2-90% N, atmosphere in arder to give a final O2

~ ~~ ~ concentration of approximately 5%. Flasks were shaken at * Corresponding author. 150 rpm for 3 to 5 days at 37°C.

31 32 PASTER ET AL. INT. J. SYST. BACTERIOL.

H. mustelae ATCC 43772 was grown as previously de- inhibition after 48 h (for C.jejunz) or 72 h (for the helicobac- scribed by Fox et al. (12). ters tested) of incubation. Isolation of H.felis strains. H.felis CSIT, CS2, CS5, and Electron microscopy. Negatively stained samples, ultrathin CS6 were isolated from the stomachs of individual cats, and sections, and freeze-etched replicas for transmission elec- strains DS1, DS2, and DS3 were isolated from the stomachs tron microscopy were prepared as previously described (28, of individual dogs. Cats and dogs were obtained from the 35). Specimens were examined by using an Hitachi model Animal Breeding and Holding Unit at the University of New 7000 transmission electron microscope. South Wales. The health status of animals was not deter- G + C content of DNA. The guanine-plus-cytocine (G + C) mined. The ages, sexes, and breeds of the animals varied. contents of DNAs were determined by thermal denaturation The animals were euthanized, and the stomachs were re- analysis as described by Breznak and Canale-Parola (3) and moved for bacterial culture. The procedures used for isola- were calculated by using the equation of De Ley (6). DNA tion and initial cultivation have been described previously isolated from K-12 was used for control (27). Briefly, mucus scrapings of gastric mucosa were determinations. streaked onto lysed horse blood agar (blood agar base no 2; Isolation and purification of rRNAs. rRNAs were isolated Oxoid, Basingstoke, United Kingdom) supplemented with and partially purified by a modification of the procedure of as described above. After 4 and 7 days of mi- Pace et al. (33), as previously described (34). croaerophilic incubation at 37"C, the plates were examined 16s rRNA sequencing. rRNA sequences were determined for spreading colonies on the agar surfaces. by using a modification of the standard Sanger dideoxy chain Biochemical characterization. Phenotypic tests commonly termination technique in which primers complementary to used to biotype campylobacters were performed, and in conserved regions of the 16s rRNA sequences were elon- some instances the procedures were modified as described gated by the reverse transcriptase (20). Seven prim- by Benjamin et al. (2). Oxidase and catalase activities were ers were used to obtain nearly complete sequences for H. assayed as described previously (2). Urease was tested by felis and H. mustelae. Additional modification of these the microtiter method as described by Hazel1 et al. (15). H,S procedures have been described previously (34). production was measured by using a modification of the Data analysis. A program set for data entry, editing, method of Skirrow and Benjamin (39). Briefly, a large sequence alignment, secondary structure comparison, simi- loopful of bacterial cells was inoculated as a lump into larity matrix generation, and phylogenetic tree construction semisolid agar deeps consisting of nutrient broth no. 2,0.6% for 16s rRNA data was written in Microsoft QuickBASIC (wthol) bacteriological agar, 0.1% (wthol) yeast extract, for use on IBM PC-AT and compatible computers. RNA 0.05% (wdvol) ferrous sulfate, 0.05% (wthol) sodium met- sequences were entered and aligned as previously described abisulfite, and 0.05% (wthol) sodium pyruvate. After 4 h of (34). Presently, our data base, which contains RNA se- incubation at 37"C, H,S production was indicated by the quences for approximately 250 different bacterial strains, presence of blackening caused by the formation of iron comprises sequences determined in our laboratory, previ- sulfide. The hippurate hydrolysis test was performed by ously published sequences, and unpublished sequences pro- using the protocol of Hwang and Ederer (16). Commercially vided by other investigators. Similarity matrices were con- available kits for enzyme analysis were utilized. The two structed by comparing only those regions that could be systems used were AN-DENT strips (API Analytab Prod- unambiguously aligned. Dendrograms were constructed by ucts, Plainview, N.Y.) and Rosco Diagnostica tablets (Rosco using the modified unweighted pair group method of Li (29). Diagnostica, Taastrup, Denmark). These kits were used GenBank accession numbers. The sequences of the micro- according to the manufacturers' instructions. Rosco Diag- organisms which we investigated are available for electronic nostica tablets were also used for determining carbohydrate retrieval from GenBank under accession numbers M37642 utilization. (for H.felis CSIT), M37643 (for H.felis DS3), and M35048 Cultures were grown under aerobic conditions, microaero- (for H. mustelae ATCC 43772). philic conditions (Oxoid type HP11 anaerobic jar equipped with a model BR56 Campylobacter gas-generating kit and RESULTS AND DISCUSSION catalyst), and anaerobic conditions (Oxoid anaerobic jar equipped with a model BR38 anaerobic gas-generating kit Several additional bacterial strains that were ultrastructur- and catalyst) at 37°C. According to the manufacturer, the use ally and physiologically similar to H. felis CSIT were iso- of a model BR56 microaerophilic gas-generating kit provides lated from the gastric mucosa of cats (strains CS2, CS5, and an oxygen concentration of about 6%. Cultures were also CS6) and dogs (strains DS1, DS2, and DS3). All of the incubated at three temperatures (25, 37, and 42°C) in a isolates were motile, gram-negative, spiral-shaped bacteria microaerophilic atmosphere. Growth on lysed horse blood which possessed periplasmic fibers that wrapped around the agar plates was determined after 72 h of incubation. cell body (Fig. 1). All strains were microaerophilic and Tolerance to 1.0% (wt/vol) glycine and tolerance to 1.5% asaccharolytic (they did not ferment glucose, maltose, man- (wt/vol) NaCl were determined by culturing organisms on nitol, lactose, ribose, and D-xylose). Key phenotypic traits lysed horse blood agar plates supplemented with each com- that differentiate H. felis from closely related bacteria are pound. The plates were incubated for 72 h under microaero- shown in Table 1. Phenotypic traits that differentiate H.felis philic conditions. from other Helicobacter species include its tightly helical Susceptibility to antimicrobial agents. The surfaces of lysed ultrastructure, its ability to reduce nitrate to nitrite, its horse blood agar plates were inoculated with a swab which resistance to nalidixic acid, its susceptibility to cephalothin, had been moistened with a heavy suspension of bacteria (ca. and its growth at 42°C (Table I). The G+C content of the 10' cells per ml). The plates were dried gently in a laminar DNA of strain CSIT was 42.5 +- 0.5 mol%. flow cabinet, and susceptibility disks (Oxoid) containing We determined approximately 95% of the total sequence nalidixic acid (30 pg) and cephalothin (30 pg) were placed for H.felis CSIT and DS3 and H. mustelae. The sequences onto the agar surfaces. Resistance to these antimicrobial of H.felis CSIT and DS3 and H. mustelae are shown in Fig. agents was determined by the absence of a clear zone of 2 aligned with the sequence of E. coli. Table 2 shows a VOL. 41, 1991 PHYLOGENY OF HELICOBACTER FELIS SP. NOV. 33

FIG. 1. Electron micrographs of pure cultures of H. felis sp. nov., showing the characteristic spiral morphology, multiple flagella and periplasmic fibers. (A) Negatively stained cell of strain DS3. Bar = 0.25 p.m. (B and C) Thin sections of strain CSIT. Bars = 0.25 p.m. (D) Freeze-etched replica of strain CSIT. Bar = 0.1 p.m. similarity matrix derived from approximately 1,200 base we investigated were divided into two major phylogenetic comparisons for the 16s rRNA sequences of H. felis CSIT groups; one group comprised the gastric bacteria (i.e., H. and DS3, H. mustelae, Campylobacter species, and phylo- felis CSIT and DS3, H. pylori, H. mustelae, “F. rappini,” genetically related species that have been described previ- and W. succinogenes), and the other group contained the ously (21, 34, 36, 40). The nearly complete sequence for remaining organisms (the true campylobacters). The average “Flexispira rappini” (34a) was included in the phylogenetic level of similarity between members of these two groups was analyses for comparative purposes. “F. rappini,” which has 84.9%. The phylogenetic relationships between these organ- not been named formally (3a), is a gram-negative organism isms and distantly related bacterial species, such as E. coli that was first isolated from aborted ovine fetuses (4, 19). A and B. fragilis, revealed average levels of similarity of only dendrogram constructed from the similarity data is shown in 76.9 and 72.0%, respectively (data not shown). Fig. 3, which is an expanded and updated version of a As Fig. 3 shows, H. felis CSIT,H. pylori, and H. mustelae phylogenetic tree published previously (34). Although only formed a cluster with an average interspecies similarity level partial sequences (approximately 860 bases [36]) of C. coli, of 94.5%. These data indicate that H. felis belongs in the C. jejuni, and C. lari were available for construction of the newly formed genus Helicobacter (13). H. felis CSIT, which dendrogram, these organisms were included in the tree to was isolated from a cat, and H. felis DS3, which was isolated illustrate their phylogenetic positions. The exact branching from a dog, were very closely related (level of similarity, positions of these organisms may change slightly when more 99.3%),indicating that these organisms are two strains of the complete sequences become available. The organisms which same species. Previous studies that demonstrated DNA 34 PASTER ET AL. INT. J. SYST.BACTERIOL.

TABLE 1. Biochemical characteristics of H.felis and related bacteria H.felis H. pylori H. mustelae W. succinogenes C.jejuni Characteristic (7 strains) NCTC 11637“ ATCC 43773“ NCTC 11488 (2 strains)b Urease (rapid) + + - Catalase + + - + Oxidase + + + + H,S production - - - + G+C content (mol%) 37 36 47 3&38 Ultrastructure Curved to spiral Straight to curved Short curved Short curved Nitrate reduction - + + + Hippurate hydrolysis - - - + Alkaline phosphatase + + - - Arginine aminopeptidase + + + + Histidine aminopeptidase + + - - Leucine aminopeptidase + + + + y-Glutamyl transpeptidase + + - + Resistance to: Nalidixic acid (30-kg disk) + (7) + - + - Cephalothin (30-pg disk) - (0) - + + + Growth in the presence of 1%Glycine + 1.5% NaCl + Growth at: 42°C + +/- + 37°C + + + 25°C - -

~~~~~~~~~~ ~ ~ ~~ ~~ ~ ~ Data from reference 13. C. jejuni strains Vic and 900. The numbers in parentheses are the numbers of H.felis strains (of the seven strains tested) that were positive. Determined by using Rosco Diagnostica tablets. Results were negative when AN-DENT strips were used. G+C content of strain CSIT. ND, Not determined.

homology between H. mustelae and H. pylori either were rRNA sequences (approximately 650 bases) of C. cinaedi inconclusive (13) or produced conflicting results (9, 12). The and C.fennelliae demonstrated that these species are related 16s rRNA sequence analysis provided definitive molecular phylogenetically to the gastric bacteria (40). When these evidence that H. mustelae is related to H. pylori and thus sequences were included in our analyses, similar results belongs the genus Helicobacter. W. succinogenes was more were obtained, although both C. cinaedi and C. fennelliae distantly related to this cluster (average level of similarity, fell outside the Helicobacter cluster (data not shown). When 91.8%). As suggested by Goodwin et al. (13), W. succino- more complete sequences of these two species become genes is sufficiently different from Helicobacter species with available, the true phylogenetic positions of these bacteria regard to phylogeny, phenotypic characteristics, can be determined. susceptibility, and biochemical traits that a separate genus Other spiral-shaped bacteria also have been observed in designation is warranted. However, on the basis of phylo- the stomachs of cats and dogs (30, 41) and nonhuman genetic data, W. succinogenes does indeed belong in the primates (5,37). These organisms have the same tight helical same family as the helicobacters. morphology as H. felis, but the periplasmic fibers are not On the basis of the results of this study, the exact present. However, like H. felis, these organisms are found phylogenetic position of “F. ruppini” is unclear. Although only in the gastric mucosa of animals. Similar bacteria have this species is most closely related to H. mustelae (level of been found in humans, and it has been suggested they are similarity, 95.5%), it is also closely related to W. succino- transmitted zoonotically to humans via animal contact (7,24, genes (level of similarity, 93.1%). These data suggest that 26). None of these has been cultured in vitro, “F. rappini” should be included in the genus Helicobacter. but isolates from cats, dogs, monkeys, and humans have However, this uncertainty may be resolved by determining been maintained in large numbers in the stomachs of labo- the phylogeny of additional unclassified organisms which ratory mice (8). have many phenotypic traits similar to those of “F. rap- The clustering of the gastric bacteria as shown in the pini.” Archer et al. (1) described microaerophilic, fusiform- dendrogram (Fig. 3) presumably has evolutionary signifi- shaped bacteria that possessed multiple bipolar flagella and a cance. It is tempting to speculate that the helicobacters and corrugated surface formed by periplasmic fibers. These related bacteria evolved in a distinct ecological habitat, such microorganisms were isolated from two humans suffering as the gastric mucus, whereas the true campylobacters and from chronic gastroenteritis. An anaerobic bacterium iso- related bacteria evolved in the large bowels and subgingival lated from the mucosal epithelium of a murine large bowel crevices. Both of these groups may have arisen from a also had a similar ultrastructure (38). This unusual ultra- common ancestor that colonized the mucous membranes of structure was also observed in spiral-shaped bacteria iso- the alimentary tracts of primitive mammals. For example, H. lated from the crypts of mice and rat gastrointestinal tracts felis strains are very closely related phylogenetically , and (35). yet they have been isolated from both cats and dogs. In previous studies, comparative analyses of partial 16s Furthermore, it is interesting that the oral species (namely, VOL. 41, 1991 PHYLOGENY OF HELICOBACTER FELIS SP. NOV. 35

cs1 .UUUAUGGAGAGUUUGAUCCUGGCUCAGAGUGAACGCUGGCGGCGUGCCUAAUACaUGCAAGUCGAACGAUGAA---GCCUAGCUUGCUAGGC---GGAUUAGuGGcGCAcGGGuGAGuAA CSl D S3 CUUUAUGGAGAGUUUGAUCCUGGCUCAGAGUGAACGCUGGCGGCGUGCCUAAUACAUGCAAGUCGAACGAUGAA---GCCUAGCUUGCUAGGU---GGAUUAGUGGCGCACGGGUGAGUAADS3 Hm .AUUAUGGAGAGUUUnAUCCUGGCUCAGAGUGAACGCUGGCGGCGUGCCUAAUACAUGCAAGUCGAACGAUGAAG-CUUCUAGCUUGCUAGAAG-UGGAUUAGUGGCGCACGGGUGAGUAA Hm Ec AAAUUGAAGAGUWGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAACGGUAACAGGAAGAAGCUUGCUUCUUUGCUGACGAGUGGCGGACGGGUGAGUAA Ec 10 20 30 40 50 60 70 a0 90 100 110 120

cs1 CGCAUAGAUGACAUGCCCWUAGUUUGGGAUAGCCACUAGAAAUGGUGA~AAUACCAAAUACUACCUACG-GGGGAAAGA------UUUA------UCGCUAAAGGAUUGGUCUAUGUC CS DS3 CGCAUAGAUAACAUGCCCWUAGUWCCGAUAGCCACUAGAAAUGGUGAUUAAUACCAAAUACUACCUACG-GGGGAAAGA------UWa------UCGCUAMGGAUUGGUCUAUGUC DS Hm CGCAUAGGWAUGUGCCCCAUAWCUGGGAUAGCCACUGGAAACGGUGAUUAAUACUGGAUACU-CCUACG-GGGgaAAGa------ua------UCGCUAUGGGAUCAGCCUAUGUC Hm EC UGUCUGGGAA-ACUGCCUGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAAC-GUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCGGAUGUGCCCAGAUG EC 130 140 150 160 170 180 190 200 210 220 230 240

cs1 CUAUCAGCUUCUUGGUGAGWAMGGCUCACnnAGGCUAUGACGGGUAUCCGGCCUGAGAGGGUGAACGGACACACUGGAACUGAGACACGGUCCAGACUCCnnCGGGAGGCaGCAGUAG CS1 DS3 CUAUCAGCUUGUUGGUGAGGUAAAGGCUCACnnACCCUAUGACGGGUAUCCGGCCUGAGAGGGUGAACGGACACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUAG DS3 Hm CUAUCAGCUUGUUGGUGAGGUaAUGGCUCACnnAGGCUaUgACGGGUAUCCGGCCUnAGAGGGUGAUCGGACACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCaGCAGUAG Hm Ec GGAUUAGCUAGUAGCUGGGGUAACGGCUCACCUAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGG Ec 250 260 2 70 280 290 300 310 320 330 340 350 360

cs1 GGAAUAUUGCUCAAUGGGCGCMGCCUGAAGCAGCAACGCCGCGUGGAGGAUGAAGGUUUUAGGAUUGUAAACUCCUUUUGUCAGAGAAGAUA------AU CSl DS3 GGAAUAUUGCUCAAUGGGCGCMGCCUgAAGCAGCAACGCCGCGUGGAGGAUGAAGGUUUUAGGAUUGUAAACUCCUUUUGUCAGAGAAGAUA------AU DS3 Hm GGAAUAUUGCUCAAUGGGCGAAAGCCUGAAGCAGCAACGCCGCGUGGAGGAUGAAGGUUUUAGGAUUGUAAACUCCUUUUCUAAGAGAAGAUA------AU Hm Ec GGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUAUGAAGAAGGCCWCGGGUUGUAAAGUACWUCAGCGGGGAGGAAGGGAGUMAG~AAUACCUUUGCUCAUU Ec 370 380 390 400 610 420 430 440 450 460 470 480

cs1 GACGWAUCUGACGMUAAGCACCGGCUAnCUCCGUGCCAGCAGCCGCCGUAAUACGGAGGGUGC~GCGUUACUCGGAAUCnCUGGGCGUAAAGAGUGCGUAGGCGGGGUUGUAAGUCA CS1 DS3 GACGGUAUCUGACGAAUMGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUACUCGGAAUCACUGGGCGUAAAGAGUGCGUAGGCGGGGUUGUAAGUCADS3 Hm GACGGUAUCUuAGGAAUAAGCACCGGC~CUCCGUGCCAGCAGCCGCGG~~ACGGAGGGUGC~GCGWACUCGGAAUCACUGGGCGUnAAGAGCGCGUAGGCGGAGUAAUAAGUCAHm EC GACGUUACCCGCAGAACAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUUUGUUAAGUCA Ec 490 500 510 520 530 540 550 560 5 70 580 590 600

CSl GAUCUGAAAUCCUAUGGCWMCCAUAGAACUGCAUUUGAAACUACAACUCUgGAGUGUGGGAGAGGUAGGUGGAAUUcUUGGUGUAGGGGUAAAAUCCGUAGAGAUCAAGAGGAAUACUCSI DS3 GAUGUGAAAUCCUAUGGCWAACCAUAGAACUGCAUUUGAAACUACAACUCUGGAGUGUGGGAGAGGUAGGUGGAAUUCUUGGUGUAGGGGUAAAAUCCGUAGAGAUCAAGAGGAAUACU DS3 Hm GAUGUGAAAUCCUGUAGCWAACUACAGAACUGCAUUUGAAACUGUUAUUCUAGAGUGUGGGAGAGGUAGGUGGAAUUCUUGGUGUAGGGGUnAAAUCCGUAGAGAUCAAGAGGAAUACU Hm Ec GAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUCUGAUACUGGCAAGCUUGAGUCUCGUAGAGGGGGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACC Ec 610 620 630 640 650 660 670 680 690 700 710 720

cs1 CAUUGCGAAGGCGACCUGCUGGAACAAUACUGACGCUGAUUGCnCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCCUAAACGAUGGAUGCUAGUUGUUGGGGGG CS1 DS3 CAUUGCGAAGGCGACCUGCUGGAACAAUACUGACGCUGAUUGCrCGAAAGCGUGGGGAGCAAACAGGAWAGAUACCCUGGUAGUCCACGCCCUAAACGAUGGAUGCUHGUUGUUGGGGGG DS3 Hm CAUUGCGAAGGCGACCUACUGGMCAUUACUGACGCUGAU-GCGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCCUAAACGAUGAAUGCUAGUUGUUGGGGUGHm Ec GGUGGCGMGGCGGCCCCCUGGACGAAGACUGACGCUCAG-GUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGUCGACUUGGAGGUUGUGCCEc 730 740 750 760 770 780 790 aoo aio a20 a30 840

cs1 CUUUGUCCUCCCAGUAAUGCAGCUAACGCCUUAAGCAUCCCGCCUGGGGAGUACGGUCGCAAGAUUAAAACUCAAAGGAAUAGACGGGGACCCGCACAAGCnnnnnnnnnngugguuuaau CS1 D S3 CWUGUCCUCCCAGUAAUGCAGCUAACGCCUUAAGCAUCCCGCCUGGGGAGUACGGUCGCAAGAWAAAACUCAAAGGAAUAGACGGGGACCCGCACAAGcGGUGGAgcAngugguuuaau DS3 Hm CW-GUCACUCCACUAAUGCAGWAACACAUUAAGCAWCCGCCUGGGGAGUACGGUCGCAAGAUUAAAACUCAAAGGAAUAGACGGGGACCCGCACAAGCGGUGGAGCAUGUGGUUUAAU Hm Ec CW-GAGGCWGGCUUCCGGAGCUAACGCGUUAAGUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAU Ec a50 860 870 880 a90 900 910 920 930 940 95 0 960

cs1 ucgammmnCGAAGAACCWACCUAGGCUUGACAUUGAAnGAAUCUGCUAGAAAUAGUGGAGUGUCuAGCUuGCUAGACCCUGAAAACAGGUGCUGCACGGCUGUCGUCAGCU CS1 DS3 wgaMnnACACGAAGAACCWACCUAGGCUUGACAUUGAAGGAAUCCCCUAGAAAUAGGGGAGuGuCuAGCuuGCUAGACCCUGAAAACAGGUGCUGCACGGCUGUCGUCAGCU DS3 Hm UCCAmnllACGCGAAGMCCWACCUAGGCUUGACAUUCAUAGAAUCUGCUAGAAAUAGCGGAGUGUCUAGWUACUAGACCUUGAAAACAGGUGCUCCACGGCUGUCGUCAGCU Hm Ec UCGAUCCMCGCGMGMCCWACCUGWCWGACAUCCACGGAAGUUUUCAGAGAUGAGAA----UGUGCCUUCGGGM-CCGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCU Ec 970 980 990 1000 1010 1020 1030 1040 1050 1060 1070

cs 1 CWCUCCUCAGAUGWGGGUUMWCCCGCAACGAGCGCMCCCUCWUCUUAGUUGCUAACAGGUAGUGCUGAGCUCUCUAAGAAUACUGCCUGCG-UAAGCAGGAGGAAGGUGAGGACG CSI DS3 CGUGUCWGAGAUGWGGGWMGUCCCGCAACGAGCGCAACCCUcWUCUUAGWGCUAACAGGUU~GCUgAGCUCUCUAAGAAUACUGCCUGCG-UAAGCAGGAGGAAGGUGAGGACG DS3 Hm CGUWCWCAGAUWUGGCUUMWCCCGCAACGAGCGCAACCCUCGUUCUUAGWGCUAGCAGWCGG-CUGAGCACUCUAAGAAGACUGCCUUCG-UnAGGAGGAGGAAGGUGAGgACG Hm Ec CCUCUUGUGAMUGWGGCUUMWCCCGCAACGAGCGCAACCCWAUCC~UGUUGCCAGCGGUCCGC-CCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUG Ec ioao 1090 1100 1110 1120 1130 1140 1150 1160 1170 1iao 1190

cs1 ACWCAAGUCAUCAUGGCCCWACGCCUAGGGCUACACACGUGCUACAAUGGGGUGCACAAAGAGAUGCAAUGCCGCGAGGUUGAGCCAAUCU-UAAAAACnnCUCUCAGUUCGGAUUGC CS1 DS3 ACGUCAAGUCAUCAUGGCCCWACGCCUACGGCUACACACGUGCUACAAUGGGGUGUACAAAGAGAUGCAAUCCCGCGAGGCUGAGCCAAUCU-UAAAAACAUCUCUCAGUUCGGAUUGC DS3 Hm ACWUAAGUCAUCAUGCCCCWACGCCUAGGGCUACACACGUGCUACAAUGGGGUGCACAAAGAGACGCAAUACCGCGACGUGGAGCAAAUCU-CAAAAACAUCUCUCAGUUCGGAWGU Hm EC ACGUCAAGUCAUCAUGGCCCWACGACCAGGGCUACACACGUGCUACAAUGGCGCAUACAMGAGAAGCGACCUCGCGAGAGCAAGCGGACCUCAUAAAGUGCGUCGUAGUCCGGAUUGG Ec 1200 1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310

cs1 AGGCUGCMCUCGCCUGCAUGAAGCUGGAAUCGCUAGUAAUCGCAAAUCAGCCAUGUUGCGGUGAAUACGUUCCCGGGUCUUGUACUCACCGnnCGUCACACCAUGGGAGUUGUGUUUGCCCS1 D S3 AGGCUGCAACUCGCCUGCAUGAAGCUGGAAUCGCUAGUAAUCGCAAAUCAGCCAUGUUGCGGUGAAUACGUUCCCGGGUCUUGUACUCACCGnnCGUCACACCAUGGGAGUUGUGUUUGCCDS3 Hm AWCUGCAACUCGACUACAUGAAGCUGGAAUCGCUAGUAAUCGUGAAUCAGCCAUGUCACGGUgAAUACGUUCCCGGGUCUUGUACUCACCGnCCGUCACACCAUGGGAGUUGUAUUCGCCHm Ec AWCUGCAACUCGACUCCAUGAAWCGGAAUCGCUA~AAUCGUGGAUCAG-AAUGCCACGGUGAAUACGUUCCCGGCCCWGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGC~ EC 1320 1330 1340 1350 1360 1370 1380 1390 1400 1410 1420 1430

cs1 UUAAGUCAGGAUGCUM------GGUAGCUACUGCCCACGGCACACACAGCGACUGGG G...... CS1 DS3 UUMGUCACGAUGCUM------AGUAGCUACUGCCCACGCCACACACAGCGACUGGGG ...... DS3 Hm UUMCCCGGGAUGCUM------AWGGCUACCGUCCAnCGCGGAUnC...... Hm EC AGAAGUAGGUAGCWAACCWCGGGAGGGCGCUUACCACUWGUGAUUCAUGACUCGGGUGAACUCGUAACAAGGUAACCGUAGGGGAACCUGCCGWGGAUCACCUCCUUA EC 1440 1450 1460 1470 1Lao 1490 1500 1510 1520 1530 1540 FIG. 2. Sequences of H.felis CSIT and DS3 and H. mustelue (Hm)aligned with the sequence of E. coli (Ec). Numbering is relative to E. coli base positions. A and a, Adenine; C and c, cytosine; G and g, guanine; U and u, uracil; n, base could not be determined. Lower-case letters indicate some uncertainty in base identification. Dashes indicate gaps that were inserted for sequence alignment, and dots indicate regions that were not sequenced. 36 PASTER ET AL. INT. J. SYST.BACTERIOL.

TABLE 2. Similarity matrixa

CS1 DS3 Hp Hm Fr Ws Bg Wr Wc Cc Cf Ch Ci Cj C1 Bu Cs csi - 99.3 95.9 94.0 91.9 90.1 82.0 82.4 84.9 83.6 84.3 82.6 82.3 83.0 83.3 81.9 84.2 DS3 0.8 - 95.7 93.6 92.1 90.3 82.4 82.6 85.1 83.8 84.3 82.6 82.3 83.0 83.3 81.9 84.2 Hp 4.2 4.4 - 93.7 92.9 90.5 83.1 83.7 86.2 84.6 84.8 83.4 83.8 84.1 83.8 82.7 85.3 Hm 6.3 6.7 6.1 - 95.5 93.3 83.5 84.4 86.8 85.2 85.8 84.3 85.3 85.6 86.0 84.0 86.9 Fr 8.5 8.3 7.5 4.6 - 93.1 85.1 85.7 87.4 86.8 87.2 85.5 86.6 85.9 86.1 85.6 80.2 WS 10.6 10.4 10.1 7.0 7.3 - 84.6 85.6 87.8 85.5 85.9 84.1 85.3 85.4 85.1 84.8 86.3 Bg 20.6 20.5 19.1 18.6 16.6 17.3 - 95.6 95.0 94.4 94.1 93.1 93.0 92.2 91.5 92.2 94.3 Wr 20.1 20.0 18.4 17.5 15.9 16.0 4.5 - 96.4 95.8 94.6 93.0 94.1 93.0 92.3 93.3 94.8 Wc 16.8 16.6 15.3 14.5 13.8 13.3 5.2 3.7 - 97.0 95.6 94.2 94.8 93.6 92.8 92.3 95.1 CC 18.5 18.5 17.3 16.4 14.5 16.1 5.8 4.3 3.0 - 96.0 94.1 94.9 94.4 94.4 92.6 94.9 Cf 17.6 17.6 17.0 15.8 14.0 15.7 6.1 5.6 4.6 4.1 - 98.2 96.0 94.9 94.4 93.7 94.7 Ch 19.9 19.9 18.8 17.6 16.1 17.9 7.3 7.4 6.0 6.1 1.8 - 96.9 95.7 94.8 92.7 93.4 Ci 20.2 20.2 18.2 16.4 14.8 16.4 7.3 6.1 5.4 5.3 4.1 3.1 - 97.8 96.5 92.9 93.8 Cj 19.3 19.3 17.8 15.9 15.6 16.3 8.2 7.4 6.7 5.8 5.3 4.5 2.2 - 97.8 92.4 92.8 C1 18.9 18.9 18.3 15.5 15.3 16.6 9.0 8.2 7.5 5.8 5.9 5.4 3.6 2.2 - 92.2 92.7 Bu 20.8 20.8 19.7 18.0 16.0 17.0 8.2 7.0 8.2 7.8 6.6 7.7 7.4 8.0 8.2 - 92.9 CS 17.8 17.8 16.3 14.4 12.8 15.1 5.9 5.4 5.1 5.3 5.5 7.0 6.5 7.5 7.6 7.4 - --_----___--______------__---______------

a Abbreviations: CS1, H. felis CSIT; DS3, H. felis DS3; Hp, H. pylori; Hm, Hm, H. mustelae; Fr, “F. rappini”; Ws, W. succinogenes; Bg, B. gracilis; Wr, W. recta; Wc, W.curva; Cc, C. concisus; Cf, C.fetus subsp. fetus; Ch, C. hyointestinalis; Ci, C. coli; Cj, C.jejuni; C1, C. lari; Bu, B. ureolyticus;Cs, C. sputorum subsp. bubulus. Sequences for “F. rappini” 1937, C.fetus subsp. fetus ATCC 27374, and C. hyointestinalis ATCC 35217 are unpublished data. Other previously published sequences were obtained elsewhere (34, 36). The numbers above the diagonal represent uncorrected percentages of similarity. The numbers below the diagonal are percentages of difference corrected for multiple base changes by the method of Jukes and Cantor (18).

W. recta, W. curva, C. concisus, and B. gracilis) form a tight (diameter, 2 to 4 pm) are present in older cultures. No subcluster within the true campylobacters (Fig. 3). endospores are produced. Cells are motile with a rapid Description of Helicobacter felis sp. nov. Helicobacter felis corkscrewlike motion. Cells have tufts of 10 to 17 polar (fe’ lis. L. gen. n. felis, of a cat). Rigid, spiral-shaped, sheathed flagella (thickness, 25 nm) that are positioned gram-negative cells that are 0.4 pm wide and 5 to 7.5 p,m slightly off center at the end of the cell. Cells are surrounded long and have five to seven spirals per cell. Spherical forms by periplasmic fibers which appear as concentric helical

0.05 Helfcobacter fells CSJ He1 f cobact er f e 1f s 053 Helicobacter pylorf Helfcobacter mustelae ’Flexispfra rappinf Wolinella succinogenes Bacterofdes gracilis ir’olfnella recta Campy1obacter concisus ir’olfnella curva Camp ylobac ter sputorum ss bubulus 1 Campylobacter fetus ss fetus I Campylobac t er hyo n test nal c f f i s - Camp ylobac t er colf Campylobacter fejuni - Camp ylobac ter larf Bacteroides ureolyticus FIG. 3. Phylogenetic tree for the organisms analyzed in Table 2. The scale bar represents a 5% difference in nucleotide sequence as determined by taking the sum of all of the horizontal lines connecting two species. Vertical distance has no meaning. VOL. 41. 1991 PHYLOGENY OF HELZCOBACTER FELZS SP. NOV. 37 ridges, either in pairs, threes, or singly on the surfaces of the Med. Microbiol. 2955-62. cells. Microaerophilic, but can grow anaerobically. Grows at 9. Fox, J. G., T. Chilvers, C. S. Goodwin, N. S. Taylor, P. 37 and 42°C but not at 25°C. One strain does not grow at Edmonds, L. I. Sly, and D. J. Brenner. 1989. Campylobacter 42°C. Nutritionally fastidious, growing only on media en- mustelae, a new species resulting from the elevation of Cam- riched with blood or serum. Asaccharolytic. No acid is pyfobacterpyfori subsp. mustelae to species status. Int. J. Syst. Bacteriol. 39:301-303. produced from maltose, sucrose, lactose, fructose, xylose, 10. Fox, J. G., P. Correa, N. S. Taylor, A. Lee, G. Otto, J. C. sorbitol, arabinose, raffinose, glucose, and galactose. Ure- Murphy, and R. Rose. 1990. Helicobacter mustelae-associated ase, oxidase, and catalase positive. Alkaline phosphatase, gastritis in ferrets: an animal model of Helicobacter pylori arginine aminopeptidase, leucine aminopeptidase, and y-glu- gastritis in humans. Gastroenterology 99:352-361. tamyl transpeptidase activities are detected. Most strains 11. Fox, J. G., and A. Lee. 1989. Gastric campylobacter-like organ- have histidine and leucine aminopeptidase activity. No pro- isms: their role in gastric disease in laboratory animals. Lab. duction of N-acetylglucosaminidase, a-glucosidase , a-arab- Anim. Sci. 39543-553. inosidase, P-glucosidase, a-fucosidase, a-galactosidase, 12. Fox, J. G., N. S. Taylor, P. Edmonds, and D. J. Brenner. 1988. P-galactosidase, indoxylacetate, proline aminopeptidase, Campylobacter pylori subsp. mustelae subsp. nov. isolated from the gastric mucosa of ferrets (Mustela putorius furo), and pyroglutamic acid amylamidase, tyrosine aminopeptidase, an emended description of Campylobacter pyfori. Int. J. Syst. alanine aminopeptidase, phenylalanine aminopeptidase, gly- Bacteriol. 38:367-370. cine aminopeptidase, and arginine dihydrolase. Nitrate is 13. Goodwin, C. S., J. A. Armstrong, T. Chilvers, M. Peters, M. D. reduced to nitrite. Hippurate is not hydrolyzed. Indole and Collins, L. Sly, W. McConnell, and W. E. S. Harper. 1989. H,S are not produced. No growth occurs in the presence of Transfer of Campylobacter pylori and Campylobacter mustelae 1% glycine and 1.5% NaCI. Susceptible to cephalothin, to Helicobacter pylori gen. nov. and Helicobacter mustelae ampicillin, erythromycin, metronidazole, and bismuth com- comb. nov., respectively. Int. J. Syst. Bacteriol. 39:397405. pounds, but resistant to nalidixic acid. Isolated from the 14. Graham, D. Y. 1989. Campylobacter pylori and . Gastroenterology 96:615-625. (= gastric mucosa of cats and dogs. Strain CS1 ATCC 15. Hazell, S. L., T. J. Borody, A. Gal, and A. Lee. 1987. Campylo- 49179), which was isolated from the gastric mucosa of a cat, bacter pyloridis gastritis. I. Detection of urease as a marker of is the type strain; its G+C content is 42.5 mol% (as deter- bacterial colonisation and gastritis. Am. J. Gastroenterology mined by the thermal denaturation method). 82:292-296. 16. Hwang, M. N., and G. M. Ederer. 1975. Rapid hippurate ACKNOWLEDGMENTS hydrolysis method for presumptive identification of group B streptococci. J. Clin. Microbiol. 1:114-115. We thank J. H. Bryner and I. Wesley of the National Animal 17. Jones, D. M., A. Curry, and A. J. Fox. 1985. An ultrastructural Disease Center, USDA, Ames, Iowa, for providing cell pellets of study of the gastric campylobacter-like organism “Campylobac- “F. rappini” and C.fetus subsp. fetus. Freeze-etched replicas were ter pyloridis.” J. Gen. Microbiol. 131:2335-2341. provided by S. Kouprach, UNSW Biomedical Electron Microscopy 18. Jukes, T. H., and C. R. Cantor. 1969. Evolution of protein Unit. molecules, p. 21-132. In H. N. Munro (ed.), Mammalian protein This work was supported by grant CA-26731 from the National metabolism, vol. 3. Academic Press, Inc., New York. Cancer Institute, grants AI-25631 and AI-25590 from the National 19. Kirkbride, C. A., C. E. Gates, and J. E. Collins. 1986. Abortion Institute of Allergy and Infectious Diseases, grant RR-01046 from in sheep caused by a non-classified, anaerobic, flagellated the Division of Research Resources, and grants DE-08303 and bacterium. Am. J. Vet. Res. 47:259-262. DE-04881 from the National Institute of Dental Research. 20. Lane, D. J., B. Pace, G. J. Olsen, D. A. Stahl, M. L. Sogin, and N. R. Pace. 1985. Rapid determination of 16s ribosomal RNA REFERENCES sequences for phylogenetic analyses. Proc. Natl. Acad. Sci. 1. Archer, J. R., S. Romero, A. E. Ritchie, M. E. Hamacher, B. M. USA 82: 6955-6959. Steiner, J. H. Bryner, and R. F. Schell. 1988. Characterization of 21. Lau, P. P., B. DeBrunner-Vossbrinck, B. Dunn, K. Miotto, M. T. an unclassified microaerophilic bacterium associated with gas- MacDonell, D. M. Rollins, C. J. Pillidge, R. B. Hespell, R. R. troenteritis. J. Clin. Microbiol. 26:lOl-105. Colwell, M. L. Sogin, and G. E. Fox. 1987. Phylogenetic 2. Benjamin, J., S. Leaper, R. J. Owen, and M. B. Skirrow. 1983. diversity and position of the genus Campylobacter. Syst. Appl. Description of Campylobacter laridis, a new species comprising Microbiol. 9:231-238. the nalidixic acid resistant thermophilic Campylobacter 22. Lee, A. 1984. Neglected niches, the microbiol ecology of the (NARTC) group. Curr, Microbiol. 8:231-238. . Adv. Microb. Ecol. 8:115-162. 3. Breznak, J. A., and E. Canale-Parola. 1975. Morphology and 23. Lee, A. 1989. Campylobacter pylori and CLO in animals. physiology of Spirochaeta aurantia strains isolated from aquatic Overview of mucus colonising organisms, p. 197-202. In B. habitats. Arch. Microbiol. 1051-12. Rathbone and V. Heatley (ed.), Campylobacter pylori and 3a.Bryner, J. H., J. Littleton, C. Gates, C. A. Kirkbridt, A. E. gastroduodenal disease. Blackwell Scientific Publications, Ox- Ritchie, and J. R. Archer. 1986. XIV International Congress of ford, Microbiology, Manches ter, England. 24. Lee, A. 1989. Human gastric spirilla other than C. pyfori, p. 4. Bryner, J. H., A. E. Ritchie, L. Pollet, C. A. Kirkbride, and J. E. 222-240. In M. Blaser (ed.), Campylobacter in gastritis and Collins. 1987. Experimental and abortion of pregnant peptic ulcer disease. Igaku-Shoin Medical Publishers, New guinea pigs with a unique spirillum-like bacterium isolated from York. aborted ovine fetuses. Am. J. Vet. Res. 48:91-95. 25. Lee, A., J. Dent, S. Hazell, and C. McNulty. 1988. Origin of 5. Curry, A., D. M. Jones, and J. Eldridge. 1987. Spiral organisms spiral organisms in the gastric antrum. Lancet i:300-301. (Let- in the baboon . Lancet ii:634-635. ter.) 6. De Ley, J. 1970. Reexamination of the association between 26. Lee, A., R. P. Ecksein, D. I. Fevre, E. Dick, and J. E. Kellow. melting point, buoyant density, and chemical base composition 1989. Non Campylobacter pylori spiral organisms in the gastric of deoxyribonucleic acid. J. Bacteriol. 101:738-754. antrum. Aust. N.Z. J. Med. 19:156-158. 7. Dent, J. C., C. A. M. McNulty, J. C. Uff, S. P. Wilkinson, and 27. Lee, A., and S. L. Hazell. 1988. Campylobacter pylori in health M. W. L. Gear. 1987. Spiral organisms in the gastric antrum. and disease: an ecological perspective. Microbiol. Ecol. Health Lancet ii:96. Dis. 1:l-16. 8. Dick, E., A. Lee, G. Watson, and J. O’Rourke. 1989. Use of the 28. Lee, A., S. L. Hazell, J. O’Rourke, and S. Kouprach. 1988. mouse for the isolation and investigation of stomach-associated Isolation of a spiral-shaped bacterium from the cat stomach. spiral-helical shaped bacteria from man and other animals. J. Infect. Immun. 56:2843-2850. 38 PASTER ET AL. INT. J. SYST.BACTERIOL.

29. Li, W.-H. 1981. Simple method for constructing phylogenetic 34a.Paster, B. J., and F. E. Dewhirst. Unpublished data. trees from distance matrices. Proc. Natl. Acad. Sci. USA 35. Phillips, M. W., and A. Lee. 1983. Isolation and characterization 78: 1085-1089. of a spiral bacterium from the crypts of rodent gastrointestinal 30. Lockard, V. G., and R. K. Boler. 1970. Ultrastructure of a tracts. Appl. Environ. Microbiol. 45:675-683. spiraled microorganism in the gastric mucosa of dogs. Am. J. 36. Romaniuk, P. J, B. Zoltowska, T. J. Trust, D. J. Lane, G. J. Vet. Res. 31:1453-1462. Olsen, N. R. Pace, and D. A. Stahl. 1987. Campylobacter pyfori, 31. Marshall, B. J., H. Royce, D. 1. Annear, C. S. Goodwin, J. W. the spiral bacterium associated with human gastritis, is not a Pearman, J. R. Warren, and J. A. Armstrong. 1984. Original true Campylobacter sp. J. Bacteriol. 169:2137-2141. isolation of Campylobacter pyloridis from human gastric mu- 37. Sato, T., and A. Takeuchi. 1982. Infection by spirilla in the cosa. Microbios Lett. 2583-88. stomach of the rhesus monkey. Vet. Pathol. 19:17-25. 32. Morris, G. K., and C. M. Patton. 1985. Campylobacter, p. 302-308. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and 38. Savage, D. C., J. S. McAllister, and C. P. Davis. 1971. Anaerobic H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. bacteria on the mucosal epithelium of the murine large bowel. American Society for Microbiology, Washington, D.C. Infect. Immun. 4:492-502. 33. Pace, B., E. A. Matthews, K. D. Johnson, C. R. Cantor, and 39. Skirrow, M. B., and J. Benjamin. 1980. Differentiation of N. R. Pace. 1982. Conserved 5s rRNA complement to tRNA is enteropathogenic campylobacters. J. Clin. Pathol. 33:1122. not required for protein synthesis. Proc. Natl. Acad. Sci. USA 40. Thompson, L. M., 111, R. M. Smibert, J. L. Johnson, and N. R. 79:36-40. Krieg. 1988. Phylogenetic study of the genus Campylobacter. 34. Paster, B. J., and F. E. Dewhirst. 1988. Phylogeny of campylo- Int. J. Syst. Bacteriol. 38:19&200. bacters, wolinellas, Bacteroides gracilis, and Bacteroides ure- 41. Weber, A. F., and E. F. Schmittdiel. 1962. Electron microscopic olyticus by 16s ribosomal ribonucleic acid sequencing. Int. J. and bacteriologic studies of spirilla isolated from the fundic Syst. Bacteriol. 38:56-62. stomach of cats and dogs. Am. J. Vet. Res. 23:422-427.