INTERNATIONALJOURNALOF SYSTEMATIC
BACTERIOLOGY,
Jan. 1991, p. 31-38
0020-7713/91/01003 1-08$02.00/0
Copyright 0 1991, International Union of Microbiological Societies
Vol. 41, No. 1
Phylogeny of Helicobacter felis sp. nov. , Helicobacter mustelae,
and Related Bacteria
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, urease-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 species of campylobacters. 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 campylobacters. 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 genus Helicobacter, for which we propose the name Helicobacter felis.
have their own distinctive gastric floras (11, 23). A gramnegative, microaerophilic, urease-positive, spiral-shaped rod
(strain CSIT [T = type strain]) was isolated from the gastric
mucosa of a cat and has been described previously (28).
Strain CSIT is physiologically similar to, but morphologically different from, H. pylori and H . mustelae. The gastric
bacteria isolated thus far have different distinctive morphologies with respect to cell shape and number and arrangement
of flagella (11, 17).
In this study, six additional bacterial strains were isolated
from the gastric mucosa of dogs and cats. These isolates
were similar to strain CSIT with respect to ultrastructural
and biochemical characteristics. The partial 1 6 s rRNA sequences for the cat gastric spirillum (strain CSIT)and for one
dog isolate (strain DS3) were determined in order to establish their phylogenetic position among the campylobacters,
H. pylori, and related bacteria. As discussed below, the
name Helicobacter felis is proposed for these spirillumlike
organisms, and we refer to them below by the proposed
name, In addition, we present the 16s rRNA sequence of H.
mustelae since the phylogenetic relationship of this organism
to other bacteria was uncertain previously.
Cumpylobacter species commonly colonize the alimentary
and genital tracts of mammals and birds, and some are
responsible for gastrointestinal diseases (32). It has been
proposed that the mucus lining the gut mucosa is the
ecological niche inhabited by these bacteria (22, 25). Recently, increased attention has been focused on the species
isolated from the stomachs of mammals, including humans
(14,24). The gastric mucus appears to be the natural habitat
of Helicobacter pylori, a bacterium associated with histological gastritis in human stomachs (27, 31), and Helicobacter
mustelae, an organism associated with gastritis and ulcers in
adult ferrets (9, 10). On the basis of 16s rRNA sequencing
data and biochemical and phenotypic criteria, these two
species were recently transferred from the genus Campylobacter to the genus Helicobacter (13). The overall phylogeny
of members of the genus Campylobacter and related bacteria
has been studied extensively (21, 34, 36, 40). These bacteria
fall into three phylogenetic groups. The first group comprises
the true campylobacters, including Campylobacter jejuni,
subspecies of Campylobacter fetus, Campylobacter hyointestinalis, Campy lobacter concisus, Campylobacter mucosalis, Campylobacter sputorum, Campylobacter coli,
Campylobacter lari, Wolinella recta, Wolinella curva, and
two misclassified bacteroides, Bacteroides gracilis and Bacteroides ureoiyticus. The second group contains H. pylori,
H. mustelae, Wolinella succinogenes, and two misclassified
campylobacters, Campylobacter cinaedi and Campylobacter fennelliae (13, 40; unpublished data). A third group,
which presently consists of only two species, Campylobacter cryaerophila and Campylobacter nitrofigilis, branches o f f
from the other two groups at a deeper phylogenetic level
MATERIALS AND METHODS
Bacterial strains and culture conditions. Strains of H. felis
were grown in brain heart infusion broth (Difco Laboratories, Detroit, Mich.) supplemented with 5% sterile fetal calf
serum (Sigma Chemical Co., St. Louis, Mo.) and an antibiotic combination consisting of vancomycin (10 ng/ml), polymycin (2.5 pg/ml), trimethoprim (5 pg/ml), and amphotericin
(2 kg/ml). Flasks containing liquid media were evacuated
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
150 rpm for 3 to 5 days at 37°C.
(40).
Many animal species, such as dogs, cats, and primates,
~
~~
~
* Corresponding author.
31
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32
PASTER ET AL.
INT.
H . mustelae ATCC 43772 was grown as previously described by Fox et al. (12).
Isolation of H . felis strains. H . felis CSIT, CS2, CS5, and
CS6 were isolated from the stomachs of individual cats, and
strains DS1, DS2, and DS3 were isolated from the stomachs
of individual dogs. Cats and dogs were obtained from the
Animal Breeding and Holding Unit at the University of New
South Wales. The health status of animals was not determined. The ages, sexes, and breeds of the animals varied.
The animals were euthanized, and the stomachs were removed for bacterial culture. The procedures used for isolation and initial cultivation have been described previously
(27). Briefly, mucus scrapings of gastric mucosa were
streaked onto lysed horse blood agar (blood agar base no 2;
Oxoid, Basingstoke, United Kingdom) supplemented with
antibiotics as described above. After 4 and 7 days of microaerophilic incubation at 37"C, the plates were examined
for spreading colonies on the agar surfaces.
Biochemical characterization. Phenotypic tests commonly
used to biotype campylobacters were performed, and in
some instances the procedures were modified as described
by Benjamin et al. (2). Oxidase and catalase activities were
assayed as described previously (2). Urease was tested by
the microtiter method as described by Hazel1 et al. (15). H,S
production was measured by using a modification of the
method of Skirrow and Benjamin (39). Briefly, a large
loopful of bacterial cells was inoculated as a lump into
semisolid agar deeps consisting of nutrient broth no. 2,0.6%
(wthol) bacteriological agar, 0.1% (wthol) yeast extract,
0.05% (wdvol) ferrous sulfate, 0.05% (wthol) sodium metabisulfite, and 0.05% (wthol) sodium pyruvate. After 4 h of
incubation at 37"C, H,S production was indicated by the
presence of blackening caused by the formation of iron
sulfide. The hippurate hydrolysis test was performed by
using the protocol of Hwang and Ederer (16). Commercially
available kits for enzyme analysis were utilized. The two
systems used were AN-DENT strips (API Analytab Products, Plainview, N.Y.) and Rosco Diagnostica tablets (Rosco
Diagnostica, Taastrup, Denmark). These kits were used
according to the manufacturers' instructions. Rosco Diagnostica tablets were also used for determining carbohydrate
utilization.
Cultures were grown under aerobic conditions, microaerophilic conditions (Oxoid type HP11 anaerobic jar equipped
with a model BR56 Campylobacter gas-generating kit and
catalyst), and anaerobic conditions (Oxoid anaerobic jar
equipped with a model BR38 anaerobic gas-generating kit
and catalyst) at 37°C. According to the manufacturer, the use
of a model BR56 microaerophilic gas-generating kit provides
an oxygen concentration of about 6%. Cultures were also
incubated at three temperatures (25, 37, and 42°C) in a
microaerophilic atmosphere. Growth on lysed horse blood
agar plates was determined after 72 h of incubation.
Tolerance to 1.0% (wt/vol) glycine and tolerance to 1.5%
(wt/vol) NaCl were determined by culturing organisms on
lysed horse blood agar plates supplemented with each compound. The plates were incubated for 72 h under microaerophilic conditions.
Susceptibility to antimicrobial agents. The surfaces of lysed
horse blood agar plates were inoculated with a swab which
had been moistened with a heavy suspension of bacteria (ca.
10' cells per ml). The plates were dried gently in a laminar
flow cabinet, and susceptibility disks (Oxoid) containing
nalidixic acid (30 pg) and cephalothin (30 pg) were placed
onto the agar surfaces. Resistance to these antimicrobial
agents was determined by the absence of a clear zone of
J.
SYST.
BACTERIOL.
inhibition after 48 h (for C . jejunz) or 72 h (for the helicobacters tested) of incubation.
Electron microscopy. Negatively stained samples, ultrathin
sections, and freeze-etched replicas for transmission electron microscopy were prepared as previously described (28,
35). Specimens were examined by using an Hitachi model
7000 transmission electron microscope.
G + C content of DNA. The guanine-plus-cytocine (G + C)
contents of DNAs were determined by thermal denaturation
analysis as described by Breznak and Canale-Parola (3) and
were calculated by using the equation of De Ley (6). DNA
isolated from Escherichia coli K-12 was used for control
determinations.
Isolation and purification of rRNAs. rRNAs were isolated
and partially purified by a modification of the procedure of
Pace et al. (33), as previously described (34).
16s rRNA sequencing. rRNA sequences were determined
by using a modification of the standard Sanger dideoxy chain
termination technique in which primers complementary to
conserved regions of the 16s rRNA sequences were elongated by the enzyme reverse transcriptase (20). Seven primers were used to obtain nearly complete sequences for H .
felis and H . mustelae. Additional modification of these
procedures have been described previously (34).
Data analysis. A program set for data entry, editing,
sequence alignment, secondary structure comparison, similarity matrix generation, and phylogenetic tree construction
for 16s rRNA data was written in Microsoft QuickBASIC
for use on IBM PC-AT and compatible computers. RNA
sequences were entered and aligned as previously described
(34). Presently, our data base, which contains RNA sequences for approximately 250 different bacterial strains,
comprises sequences determined in our laboratory, previously published sequences, and unpublished sequences provided by other investigators. Similarity matrices were constructed by comparing only those regions that could be
unambiguously aligned. Dendrograms were constructed by
using the modified unweighted pair group method of Li (29).
GenBank accession numbers. The sequences of the microorganisms which we investigated are available for electronic
retrieval from GenBank under accession numbers M37642
(for H . felis CSIT), M37643 (for H . felis DS3), and M35048
(for H . mustelae ATCC 43772).
RESULTS AND DISCUSSION
Several additional bacterial strains that were ultrastructurally and physiologically similar to H . felis CSIT were isolated from the gastric mucosa of cats (strains CS2, CS5, and
CS6) and dogs (strains DS1, DS2, and DS3). All of the
isolates were motile, gram-negative, spiral-shaped bacteria
which possessed periplasmic fibers that wrapped around the
cell body (Fig. 1). All strains were microaerophilic and
asaccharolytic (they did not ferment glucose, maltose, mannitol, lactose, ribose, and D-xylose). Key phenotypic traits
that differentiate H . felis from closely related bacteria are
shown in Table 1. Phenotypic traits that differentiate H . felis
from other Helicobacter species include its tightly helical
ultrastructure, its ability to reduce nitrate to nitrite, its
resistance to nalidixic acid, its susceptibility to cephalothin,
and its growth at 42°C (Table I). The G+C content of the
DNA of strain CSIT was 42.5 +- 0.5 mol%.
We determined approximately 95% of the total sequence
for H . felis CSIT and DS3 and H . mustelae. The sequences
of H . felis CSIT and DS3 and H . mustelae are shown in Fig.
2 aligned with the sequence of E . coli. Table 2 shows a
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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
comparisons for the 16s rRNA sequences of H. felis CSIT
and DS3, H. mustelae, Campylobacter species, and phylogenetically related species that have been described previously (21, 34, 36, 40). The nearly complete sequence for
“Flexispira rappini” (34a) was included in the phylogenetic
analyses for comparative purposes. “ F . rappini,” which has
not been named formally (3a), is a gram-negative organism
that was first isolated from aborted ovine fetuses (4, 19). A
dendrogram constructed from the similarity data is shown in
Fig. 3, which is an expanded and updated version of a
phylogenetic tree published previously (34). Although only
partial sequences (approximately 860 bases [36]) of C. coli,
C. jejuni, and C . lari were available for construction of the
dendrogram, these organisms were included in the tree to
illustrate their phylogenetic positions. The exact branching
positions of these organisms may change slightly when more
complete sequences become available. The organisms which
we investigated were divided into two major phylogenetic
groups; one group comprised the gastric bacteria (i.e., H.
felis CSIT and DS3, H. pylori, H . mustelae, “ F . rappini,”
and W. succinogenes), and the other group contained the
remaining organisms (the true campylobacters). The average
level of similarity between members of these two groups was
84.9%.The phylogenetic relationships between these organisms and distantly related bacterial species, such as E. coli
and B . fragilis, revealed average levels of similarity of only
76.9 and 72.0%, respectively (data not shown).
As Fig. 3 shows, H. felis CSIT,H . pylori, and H . mustelae
formed a cluster with an average interspecies similarity level
of 94.5%. These data indicate that H. felis belongs in the
newly formed genus Helicobacter (13). H. felis CSIT, which
was isolated from a cat, and H. felis DS3, which was isolated
from a dog, were very closely related (level of similarity,
99.3%),indicating that these organisms are two strains of the
same species. Previous studies that demonstrated DNA
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34
INT. J. SYST.BACTERIOL.
PASTER ET AL.
TABLE 1. Biochemical characteristics of H . felis and related bacteria
Characteristic
Urease (rapid)
Catalase
Oxidase
H,S production
G+C content (mol%)
Ultrastructure
Nitrate reduction
Hippurate hydrolysis
Alkaline phosphatase
Arginine aminopeptidase
Histidine aminopeptidase
Leucine aminopeptidase
y-Glutamyl transpeptidase
Resistance to:
Nalidixic acid (30-kg disk)
Cephalothin (30-pg disk)
Growth in the presence of
1%Glycine
1.5% NaCl
Growth at:
42°C
37°C
25°C
~~~~~~~~~~
H . felis
(7 strains)
H . pylori
NCTC 11637“
H . mustelae
ATCC 43773“
W . succinogenes
NCTC 11488
+
+
+
+
+
+
-
36
Straight to curved
-
37
Curved to spiral
+
+
+
+
+
+
-
-
+
+
+
+
+
+
+ (7)
-
+
-
- (0)
+
+
-
~
~
~~
~~
~
C . jejuni
(2 strains)b
-
+
-
+
+
+
47
Short curved
3&38
Short curved
-
-
+-
+
+
-
+
+
+/-
+
+
+
++
+
-
+
+
+
+
+
-
~
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
inconclusive (13) or produced conflicting results (9, 12). The
16s rRNA sequence analysis provided definitive molecular
evidence that H . mustelae is related to H . pylori and thus
belongs the genus Helicobacter. W. succinogenes was more
distantly related to this cluster (average level of similarity,
91.8%). As suggested by Goodwin et al. (13), W. succinogenes is sufficiently different from Helicobacter species with
regard to phylogeny, phenotypic characteristics, antibiotic
susceptibility, and biochemical traits that a separate genus
designation is warranted. However, on the basis of phylogenetic data, W. succinogenes does indeed belong in the
same family as the helicobacters.
On the basis of the results of this study, the exact
phylogenetic position of “F. ruppini” is unclear. Although
this species is most closely related to H . mustelae (level of
similarity, 95.5%), it is also closely related to W . succinogenes (level of similarity, 93.1%). These data suggest that
“ F . rappini” should be included in the genus Helicobacter.
However, this uncertainty may be resolved by determining
the phylogeny of additional unclassified organisms which
have many phenotypic traits similar to those of “ F . rappini.” Archer et al. (1) described microaerophilic, fusiformshaped bacteria that possessed multiple bipolar flagella and a
corrugated surface formed by periplasmic fibers. These
microorganisms were isolated from two humans suffering
from chronic gastroenteritis. An anaerobic bacterium isolated from the mucosal epithelium of a murine large bowel
also had a similar ultrastructure (38). This unusual ultrastructure was also observed in spiral-shaped bacteria isolated from the crypts of mice and rat gastrointestinal tracts
(35).
In previous studies, comparative analyses of partial 16s
rRNA sequences (approximately 650 bases) of C. cinaedi
and C .fennelliae demonstrated that these species are related
phylogenetically to the gastric bacteria (40). When these
sequences were included in our analyses, similar results
were obtained, although both C . cinaedi and C . fennelliae
fell outside the Helicobacter cluster (data not shown). When
more complete sequences of these two species become
available, the true phylogenetic positions of these bacteria
can be determined.
Other spiral-shaped bacteria also have been observed in
the stomachs of cats and dogs (30, 41) and nonhuman
primates (5,37). These organisms have the same tight helical
morphology as H . felis, but the periplasmic fibers are not
present. However, like H . felis, these organisms are found
only in the gastric mucosa of animals. Similar bacteria have
been found in humans, and it has been suggested they are
transmitted zoonotically to humans via animal contact (7,24,
26). None of these spiral bacteria has been cultured in vitro,
but isolates from cats, dogs, monkeys, and humans have
been maintained in large numbers in the stomachs of laboratory mice (8).
The clustering of the gastric bacteria as shown in the
dendrogram (Fig. 3) presumably has evolutionary significance. It is tempting to speculate that the helicobacters and
related bacteria evolved in a distinct ecological habitat, such
as the gastric mucus, whereas the true campylobacters and
related bacteria evolved in the large bowels and subgingival
crevices. Both of these groups may have arisen from a
common ancestor that colonized the mucous membranes of
the alimentary tracts of primitive mammals. For example, H .
felis strains are very closely related phylogenetically, and
yet they have been isolated from both cats and dogs.
Furthermore, it is interesting that the oral species (namely,
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VOL. 41, 1991
PHYLOGENY OF HELICOBACTER FELIS SP. NOV.
35
cs1
D S3
Hm
Ec
.UUUAUGGAGAGUUUGAUCCUGGCUCAGAGUGAACGCUGGCGGCGUGCCUAAUACaUGCAAGUCGAACGAUGAA---GCCUAGCUUGCUAGGC---GGAUUAGuGGcGCAcGGGuGAGuAA
CSl
CUUUAUGGAGAGUUUGAUCCUGGCUCAGAGUGAACGCUGGCGGCGUGCCUAAUACAUGCAAGUCGAACGAUGAA---GCCUAGCUUGCUAGGU---GGAUUAGUGGCGCACGGGUGAGUAADS3
.AUUAUGGAGAGUUUnAUCCUGGCUCAGAGUGAACGCUGGCGGCGUGCCUAAUACAUGCAAGUCGAACGAUGAAG-CUUCUAGCUUGCUAGAAG-UGGAUUAGUGGCGCACGGGUGAGUAA
Hm
AAAUUGAAGAGUWGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAACGGUAACAGGAAGAAGCUUGCUUCUUUGCUGACGAGUGGCGGACGGGUGAGUAA
Ec
cs1
DS3
Hm
CGCAUAGAUGACAUGCCCWUAGUUUGGGAUAGCCACUAGAAAUGGUGA~AAUACCAAAUACUACCUACG-GGGGAAAGA-------UUUA------UCGCUAAAGGAUUGGUCUAUGUC CS
CGCAUAGAUAACAUGCCCWUAGUWCCGAUAGCCACUAGAAAUGGUGAUUAAUACCAAAUACUACCUACG-GGGGAAAGA-------UWa------UCGCUAMGGAUUGGUCUAUGUC DS
CGCAUAGGWAUGUGCCCCAUAWCUGGGAUAGCCACUGGAAACGGUGAUUAAUACUGGAUACU-CCUACG-GGGgaAAGa------u a - - - - - - -UCG
- CUAUGGGAUCAGCCUAUGUC Hm
EC
UGUCUGGGAA-ACUGCCUGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAAC-GUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCGGAUGUGCCCAGAUG EC
10
130
20
140
30
150
40
160
50
170
60
180
70
190
a0
200
90
210
100
110
220
120
230
240
cs1 CUAUCAGCUUCUUGGUGAGWAMGGCUCACnnAGGCUAUGACGGGUAUCCGGCCUGAGAGGGUGAACGGACACACUGGAACUGAGACACGGUCCAGACUCCnnCGGGAGGCaGCAGUAG
DS3 CUAUCAGCUUGUUGGUGAGGUAAAGGCUCACnnACCCUAUGACGGGUAUCCGGCCUGAGAGGGUGAACGGACACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUAG
CS1
DS3
Hm
CUAUCAGCUUGUUGGUGAGGUaAUGGCUCACnnAGGCUaUgACGGGUAUCCGGCCUnAGAGGGUGAUCGGACACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCaGCAGUAG
Hm
Ec
GGAUUAGCUAGUAGCUGGGGUAACGGCUCACCUAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGG
Ec
250
260
270
280
290
300
310
320
330
340
350
360
AU
AU
GGAAUAUUGCUCAAUGGGCGAAAGCCUGAAGCAGCAACGCCGCGUGGAGGAUGAAGGUUUUAGGAUUGUAAACUCCUUUUCUAAGAGAAGAUA------------------------AU
GGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUAUGAAGAAGGCCWCGGGUUGUAAAGUACWUCAGCGGGGAGGAAGGGAGUMAG~AAUACCUUUGCUCAUU
cs1 GGAAUAUUGCUCAAUGGGCGCMGCCUGAAGCAGCAACGCCGCGUGGAGGAUGAAGGUUUUAGGAUUGUAAACUCCUUUUGUCAGAGAAGAUA------------------------DS3 GGAAUAUUGCUCAAUGGGCGCMGCCUgAAGCAGCAACGCCGCGUGGAGGAUGAAGGUUUUAGGAUUGUAAACUCCUUUUGUCAGAGAAGAUA------------------------Hm
Ec
370
380
390
400
610
420
430
440
450
460
470
CSl
DS3
Hm
Ec
480
CS1
cs1 GACGWAUCUGACGMUAAGCACCGGCUAnCUCCGUGCCAGCAGCCGCCGUAAUACGGAGGGUGC~GCGUUACUCGGAAUCnCUGGGCGUAAAGAGUGCGUAGGCGGGGUUGUAAGUCA
DS3 GACGGUAUCUGACGAAUMGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUACUCGGAAUCACUGGGCGUAAAGAGUGCGUAGGCGGGGUUGUAAGUCADS3
Hm
GACGGUAUCUuAGGAAUAAGCACCGGC~CUCCGUGCCAGCAGCCGCGG~~ACGGAGGGUGC~GCGWACUCGGAAUCACUGGGCGUnAAGAGCGCGUAGGCGGAGUAAUAAGUCAHm
EC
GACGUUACCCGCAGAACAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUUUGUUAAGUCA
490
CSl
500
510
520
530
540
550
560
570
580
590
Ec
600
GAUCUGAAAUCCUAUGGCWMCCAUAGAACUGCAUUUGAAACUACAACUCUgGAGUGUGGGAGAGGUAGGUGGAAUUcUUGGUGUAGGGGUAAAAUCCGUAGAGAUCAAGAGGAAUACUC S I
DS3 GAUGUGAAAUCCUAUGGCWAACCAUAGAACUGCAUUUGAAACUACAACUCUGGAGUGUGGGAGAGGUAGGUGGAAUUCUUGGUGUAGGGGUAAAAUCCGUAGAGAUCAAGAGGAAUACU DS3
Hm
Ec
GAUGUGAAAUCCUGUAGCWAACUACAGAACUGCAUUUGAAACUGUUAUUCUAGAGUGUGGGAGAGGUAGGUGGAAUUCUUGGUGUAGGGGUnAAAUCCGUAGAGAUCAAGAGGAAUACU
Hm
GAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUCUGAUACUGGCAAGCUUGAGUCUCGUAGAGGGGGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACC
Ec
610
cs1
DS3
Hm
Ec
620
630
640
650
660
670
680
690
700
710
720
CAUUGCGAAGGCGACCUGCUGGAACAAUACUGACGCUGAUUGCnCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCCUAAACGAUGGAUGCUAGUUGUUGGGGGG
CS1
CAUUGCGAAGGCGACCUGCUGGAACAAUACUGACGCUGAUUGCrCGAAAGCGUGGGGAGCAAACAGGAWAGAUACCCUGGUAGUCCACGCCCUAAACGAUGGAUGCUHGUUGUUGGGGGG
DS3
CAUUGCGAAGGCGACCUACUGGMCAUUACUGACGCUGAU-GCGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCCUAAACGAUGAAUGCUAGUUGUUGGGGUG
Hm
GGUGGCGMGGCGGCCCCCUGGACGAAGACUGACGCUCAG-GUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGUCGACUUGGAGGUUGUGCCEc
730
740
750
760
770
780
790
aoo
aio
a20
a30
840
cs1 CUUUGUCCUCCCAGUAAUGCAGCUAACGCCUUAAGCAUCCCGCCUGGGGAGUACGGUCGCAAGAUUAAAACUCAAAGGAAUAGACGGGGACCCGCACAAGCnnnnnnnnnngugguuuaau CS1
CWUGUCCUCCCAGUAAUGCAGCUAACGCCUUAAGCAUCCCGCCUGGGGAGUACGGUCGCAAGAWAAAACUCAAAGGAAUAGACGGGGACCCGCACAAGcGGUGGAgcAngugguuuaau DS3
D S3
Hm
Ec
CW-GUCACUCCACUAAUGCAGWAACACAUUAAGCAWCCGCCUGGGGAGUACGGUCGCAAGAUUAAAACUCAAAGGAAUAGACGGGGACCCGCACAAGCGGUGGAGCAUGUGGUUUAAU Hm
Ec
CW-GAGGCWGGCUUCCGGAGCUAACGCGUUAAGUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAU
a50
860
870
880
a90
900
910
920
930
940
95 0
960
cs1 ucgammmnCGAAGAACCWACCUAGGCUUGACAUUGAAnGAAUCUGCUAGAAAUAGUGGAGUGUCuAGCUuGCUAGACCCUGAAAACAGGUGCUGCACGGCUGUCGUCAGCU CS1
DS3
DS3 wgaMnnACACGAAGAACCWACCUAGGCUUGACAUUGAAGGAAUCCCCUAGAAAUAGGGGAGuGuCuAGCuuGCUAGACCCUGAAAACAGGUGCUGCACGGCUGUCGUCAGCU
Hm
UCCAmnllACGCGAAGMCCWACCUAGGCUUGACAUUCAUAGAAUCUGCUAGAAAUAGCGGAGUGUCUAGWUACUAGACCUUGAAAACAGGUGCUCCACGGCUGUCGUCAGCU Hm
Ec
UCGAUCCMCGCGMGMCCWACCUGWCWGACAUCCACGGAAGUUUUCAGAGAUGAGAA----UGUGCCUUCGGGM-CCGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCU
Ec
970
980
990
1000
1010
1020
1030
1040
1050
1060
1070
cs 1
DS3
Hm
Ec
CWCUCCUCAGAUGWGGGUUMWCCCGCAACGAGCGCMCCCUCWUCUUAGUUGCUAACAGGUAGUGCUGAGCUCUCUAAGAAUACUGCCUGCG-UAAGCAGGAGGAAGGUGAGGACG
CSI
CGUGUCWGAGAUGWGGGWMGUCCCGCAACGAGCGCAACCCUcWUCUUAGWGCUAACAGGUU~GCUgAGCUCUCUAAGAAUACUGCCUGCG-UAAGCAGGAGGAAGGUGAGGACG DS3
CGUWCWCAGAUWUGGCUUMWCCCGCAACGAGCGCAACCCUCGUUCUUAGWGCUAGCAGWCGG-CUGAGCACUCUAAGAAGACUGCCUUCG-UnAGGAGGAGGAAGGUGAGgACG
Hm
CCUCUUGUGAMUGWGGCUUMWCCCGCAACGAGCGCAACCCWAUCC~UGUUGCCAGCGGUCCGC-CCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUG Ec
ioao
1090
1100
1110
1120
1130
1140
1150
1160
1170
1iao
1190
cs1 ACWCAAGUCAUCAUGGCCCWACGCCUAGGGCUACACACGUGCUACAAUGGGGUGCACAAAGAGAUGCAAUGCCGCGAGGUUGAGCCAAUCU-UAAAAACnnCUCUCAGUUCGGAUUGC
DS3 ACGUCAAGUCAUCAUGGCCCWACGCCUACGGCUACACACGUGCUACAAUGGGGUGUACAAAGAGAUGCAAUCCCGCGAGGCUGAGCCAAUCU-UAAAAACAUCUCUCAGUUCGGAUUGC
CS1
DS3
Hm
EC
Ec
ACWUAAGUCAUCAUGCCCCWACGCCUAGGGCUACACACGUGCUACAAUGGGGUGCACAAAGAGACGCAAUACCGCGACGUGGAGCAAAUCU-CAAAAACAUCUCUCAGUUCGGAWGU
ACGUCAAGUCAUCAUGGCCCWACGACCAGGGCUACACACGUGCUACAAUGGCGCAUACAMGAGAAGCGACCUCGCGAGAGCAAGCGGACCUCAUAAAGUGCGUCGUAGUCCGGAUUGG
1200
cs1
DS3
Hm
Ec
1210
1220
1230
1240
1250
1260
1270
1280
1290
1300
Hm
1310
AGGCUGCMCUCGCCUGCAUGAAGCUGGAAUCGCUAGUAAUCGCAAAUCAGCCAUGUUGCGGUGAAUACGUUCCCGGGUCUUGUACUCACCGnnCGUCACACCAUGGGAGUUGUGUUUGCC
CS1
AGGCUGCAACUCGCCUGCAUGAAGCUGGAAUCGCUAGUAAUCGCAAAUCAGCCAUGUUGCGGUGAAUACGUUCCCGGGUCUUGUACUCACCGnnCGUCACACCAUGGGAGUUGUGUUUGCC
DS3
AWCUGCAACUCGACUACAUGAAGCUGGAAUCGCUAGUAAUCGUGAAUCAGCCAUGUCACGGUgAAUACGUUCCCGGGUCUUGUACUCACCGnCCGUCACACCAUGGGAGUUGUAUUCGCCHm
AWCUGCAACUCGACUCCAUGAAWCGGAAUCGCUA~AAUCGUGGAUCAG-AAUGCCACGGUGAAUACGUUCCCGGCCCWGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGC~ E C
1320
1330
1340
1350
1360
1370
1380
1390
1400
1410
1420
cs1 UUAAGUCAGGAUGCUM------GGUAGCUACUGCCCACGGCACACACAGCGACUGGG G.....................................................
DS3 UUMGUCACGAUGCUM------AGUAGCUACUGCCCACGCCACACACAGCGACUGGGG
Hm
UUMCCCGGGAUGCUM------AWGGCUACCGUCCAnCGCGGAUnC..........
EC
AGAAGUAGGUAGCWAACCWCGGGAGGGCGCUUACCACUWGUGAUUCAUGACUCGGGUGAACUCGUAACAAGGUAACCGUAGGGGAACCUGCCGWGGAUCACCUCCUUA
1440
1450
1460
1470
1Lao
1490
1500
1510
1520
1530
1540
.....................................................
......................................................
1430
CS1
DS3
Hm
EC
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.
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INT. J. SYST.BACTERIOL.
PASTER ET AL.
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
95.6
95.0
94.4
94.1
93.1
93.0
92.2
91.5
92.2
94.3
96.4
95.8
94.6
93.0
94.1
93.0
92.3
93.3
94.8
97.0
95.6
94.2
94.8
93.6
92.8
92.3
95.1
96.0
94.1
94.9
94.4
94.4
92.6
94.9
98.2
96.0
94.9
94.4
93.7
94.7
96.9
95.7
94.8
92.7
93.4
-
Bg
20.6
20.5
19.1
18.6
16.6
17.3
-
Wr
20.1
20.0
18.4
17.5
15.9
16.0
4.5
-
Wc
16.8
16.6
15.3
14.5
13.8
13.3
5.2
3.7
-
CC
18.5
18.5
17.3
16.4
14.5
16.1
5.8
4.3
3.0
-
Cf
17.6
17.6
17.0
15.8
14.0
15.7
6.1
5.6
4.6
4.1
-
Ch
19.9
19.9
18.8
17.6
16.1
17.9
7.3
7.4
6.0
6.1
1.8
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
92.2
92.7
-
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
-
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
-
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
92.9
-
-------------__---______________________------------------------------------------------------------------------------------------_----___--____________________________---------------------------------------------------------------------------------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; W r ,
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
subcluster within the true campylobacters (Fig. 3).
Description of Helicobacter felis sp. nov. Helicobacter felis
(fe’ lis. L. gen. n. felis, of a cat). Rigid, spiral-shaped,
gram-negative cells that are 0.4 pm wide and 5 to 7.5 p,m
long and have five to seven spirals per cell. Spherical forms
(diameter, 2 to 4 pm) are present in older cultures. No
endospores are produced. Cells are motile with a rapid
corkscrewlike motion. Cells have tufts of 10 to 17 polar
sheathed flagella (thickness, 25 nm) that are positioned
slightly off center at the end of the cell. Cells are surrounded
by periplasmic fibers which appear as concentric helical
0.05
Helfcobacter f e l l s CSJ
He1f cobact e r f e 1f s 053
Helicobacter p y l o r f
Helfcobacter mustelae
’ F l e x i s p f r a rappinf
W o l i n e l l a succinogenes
Bacterofdes g r a c i l i s
ir’olfnella r e c t a
1I
Campy1obacter concisus
ir’olfnella curva
Campylobac t e r sputorum ss bubulus
Campylobacter fetus ss f e t u s
c
-
-
Campylobac t e r hyof n t e s t f nal is
Campylobac t e r c o l f
Campylobacter f e j u n i
Campylobac t e r l a r f
Bacteroides u r e o l y t i c u s
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.
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VOL. 41. 1991
PHYLOGENY OF HELZCOBACTER FELZS SP. NOV.
ridges, either in pairs, threes, or singly on the surfaces of the
cells. Microaerophilic, but can grow anaerobically. Grows at
37 and 42°C but not at 25°C. One strain does not grow at
42°C. Nutritionally fastidious, growing only on media enriched with blood or serum. Asaccharolytic. No acid is
produced from maltose, sucrose, lactose, fructose, xylose,
sorbitol, arabinose, raffinose, glucose, and galactose. Urease, oxidase, and catalase positive. Alkaline phosphatase,
arginine aminopeptidase, leucine aminopeptidase, and y-glutamyl transpeptidase activities are detected. Most strains
have histidine and leucine aminopeptidase activity. No production of N-acetylglucosaminidase, a-glucosidase , a-arabinosidase, P-glucosidase, a-fucosidase, a-galactosidase,
P-galactosidase, indoxylacetate, proline aminopeptidase,
pyroglutamic acid amylamidase, tyrosine aminopeptidase,
alanine aminopeptidase, phenylalanine aminopeptidase, glycine aminopeptidase, and arginine dihydrolase. Nitrate is
reduced to nitrite. Hippurate is not hydrolyzed. Indole and
H,S are not produced. No growth occurs in the presence of
1% glycine and 1.5% NaCI. Susceptible to cephalothin,
ampicillin, erythromycin, metronidazole, and bismuth compounds, but resistant to nalidixic acid. Isolated from the
gastric mucosa of cats and dogs. Strain CS1 (= ATCC
49179), which was isolated from the gastric mucosa of a cat,
is the type strain; its G+C content is 42.5 mol% (as determined by the thermal denaturation method).
ACKNOWLEDGMENTS
We thank J. H. Bryner and I. Wesley of the National Animal
Disease Center, USDA, Ames, Iowa, for providing cell pellets of
“ F . rappini” and C .fetus subsp. fetus. Freeze-etched replicas were
provided by S . Kouprach, UNSW Biomedical Electron Microscopy
Unit.
This work was supported by grant CA-26731 from the National
Cancer Institute, grants AI-25631 and AI-25590 from the National
Institute of Allergy and Infectious Diseases, grant RR-01046 from
the Division of Research Resources, and grants DE-08303 and
DE-04881 from the National Institute of Dental Research.
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