Chapter 5Taxonomy of the Helicobacter Genus

Solnick JV, Vandamme P.

Publication Details

Introduction

Background

The cultivation of a novel bacterium from gastric mucosa in 1982 marked a turning point in our understanding of gastrointestinal microbial ecology and disease. Marshall and Warren (46) described spiral or curved bacilli in histologic sections from 58 of 100 consecutive biopsies of human gastric antral mucosa. Eleven biopsies were culture positive for a gram-negative, microaerophilic bacterium, whose proper classification generated considerable interest at the Second International Workshop on Campylobacter Infections held in Brussels, Belgium, in September 1983 (57). The organism resembled Campylobacter in several respects, including curved morphology, growth on rich media under microaerophilic conditions, failure to ferment glucose, sensitivity to metronidazole, and a G + C content of 34%. It was therefore first referred to as "pyloric Campylobacter" (pylorus, Greek, gatekeeper, or one who looks both ways) and validated as Campylobacter pyloridis in 1985 (1). The specific epithet was revised to Campylobacter pylori in 1987 to conform to the correct Latin genitive of the noun pylorus (45).

Yet almost from its initial cultivation it was suspected that perhaps C. pylori was not a true Campylobacter. Early electron micrographs showed multiple sheathed flagella at one pole of the bacterium, in contrast to the single bipolar unsheathed flagellum typical of Campylobacter spp. (28). Major protein bands and fatty acids of C. pylori were also markedly different from those of Campylobacter species (28, 56). Subsequent 16S rRNA sequence analysis showed that the distance between C. pylori and the true campylobacters was sufficient to exclude it from the Campylobacter genus (60), and it was renamed Helicobacter pylori, the first member of the new genus Helicobacter (26).

Expansion of the genus Helicobacter

The cultivation of H. pylori and the recognition of its clinical significance served to renew interest in bacteria associated with the gastrointestinal and hepatobiliary tracts of humans and other animals, many of which have now been identified as novel Helicobacter species. The genus Helicobacter presently comprises 18 validly named species and two Candidatus species, a designation adopted by the International Committee on Systematic Bacteriology to record the properties of putative procaryotic taxa that are incompletely described (52). The purpose of this chapter is to describe the taxonomic characteristics of the Helicobacter genus and to discuss methods used to differentiate among Helicobacter species. We conclude with a summary of recent recommendations for the identification of novel Helicobacter species (12).

Description of the Genus Helicobacter

Cellular Morphology and Ultrastructure

Helicobacters are non-spore-forming gram-negative bacteria. The cellular morphology may be curved, spiral, or fusiform, typically 0.2 to 1.2 μm in diameter and 1.5 to 10.0 μm long. The spiral wavelength may vary with the age, the growth conditions, and the species identity of the cells. In old cultures or those exposed to air, cells may become coccoid.

Periplasmic fibers or an electron-dense glycocalyx or capsule-like layer has been observed on the cellular surface of several species (26, 43, 55, 63). Electron-dense granular bodies have been observed in H. pylori (4) and H. rodentium (63). In H. pylori these bodies are known to be aggregates of polyphosphate and may serve as a reserve energy source.

Helicobacter cells are motile, with a rapid cork-screw-like or slower wave-like motion due to flagellar activity. Strains of most species have bundles of multiple sheathed flagella with a polar or bipolar distribution. Other species have only a single polar or bipolar flagellum (Table 1). However, flagellation can be peritrichous (H. mustelae) or nonsheathed (H. pullorum, H. rodentium, and "H. mesocricetorum") as well.

Table 1. Characteristics of cultivated Helicobacter speciesa.

Table 1

Characteristics of cultivated Helicobacter speciesa.

Growth Characteristics

In laboratory conditions, strains typically grow under microaerobic conditions at 37°C. No growth is observed in aerobic conditions. H. rodentium strains grow in anaerobic as well as microaerobic conditions (63). Strains of several species do not require atmospheric hydrogen for growth in microaerobic conditions, although there may be a growth-stimulating effect of atmospheric hydrogen on the growth of such strains. However, as for some Campylobacter species (e.g., Campylobacter hyointestinalis), hydrogen requirements for microaerobic growth of Helicobacter may be strain dependent. For instance, the isolates initially described as belonging to a novel anaerobic species, "Helicobacter westmeadii," were subsequently identified as hydrogen-requiring H. cinaedi strains that grow optimally in microaerobic conditions (77). Many laboratories routinely use microaerobic conditions including hydrogen for the routine culture of Helicobacter species and the role of this atmospheric component is therefore not well documented.

Helicobacters will grow at 37°C on a variety of rich agar bases supplemented with 5% whole blood or serum. Many species require fresh media with moist agar surfaces for optimal growth conditions, though this is not usually the case for H. pylori. In such conditions, growth is often not seen as individual colonies but as a thin, sometimes colorful or watery, spreading film. The most fastidious species such as H. bizzozeronii and H. salomonis prefer very moist conditions, and a thin broth layer can be poured on top of the agar surface to stimulate growth. Prolonged incubation up to 1 week may be required.

Biochemical Characteristics

Helicobacters are chemoorganotrophs and show a respiratory type of metabolism. They are asaccharolytic when sugar catabolism is examined by standard methods (neither oxidation nor fermentation is observed). Recent studies have, however, indicated that glucose oxidation occurs in at least H. pylori (6, 72) (see chapter 10). The glycolysis-gluconeogenesis pathway probably comprises the principal means of energy production as well as the starting point for many biosynthetic pathways. The Entner-Doudoroff pathway, the pentose phosphate shunt, and the tricarboxylic acid cycle are at least partially present; the glyoxylate shunt is absent (6, 72).

Gelatin, starch, casein, and tyrosine are not hydrolyzed. Helicobacters are methyl red and Voges-Proskauer negative. Oxidase activity is present in all species. Strains of most species produce catalase. Many species produce urease, alkaline phosphatase, or both. There is no production of pigments.

Other Characteristics

The moles percent G + C range of the DNA ranges from 30 (H. acinonychis) to 48 (H. canis). One species, H. nemestrinae, was reported to have a DNA base ratio of 24% (5). However, repeat analyses of the H. nemestrinae type strain yielded a reproducible value of 39 mol% (P. Vandamme, unpublished observations). It would therefore be appropriate to do a comparative analysis of various subcultures of the H. nemestrinae type strain to verify the strain identity in the different public and private culture collections.

Internal transcribed spacers or intervening sequences have been described in the 16S ribosomal RNA genes of H. canis (44), H. bilis (19), "H. typhlonicus" (24), and in an unnamed helicobacter referred to as Helicobacter sp. cotton-top (61). Similar intervening sequences have also been found in the 23S rRNA gene of H. canis, H. mustelae, and H. muridarum (34). The length of intervening sequences in the 16S rRNA gene ranges from 187 bp to 235 bp, while those found in the 23S rRNA gene range from 93 bp to 377 bp.

The cellular fatty acid components have been studied for a restricted number of Helicobacter species. Major cellular fatty acids reported include tetradecanoic acid; most species contain moderate to high levels of hexadecanoic acid and octadecanoic acid (27, 41).

The isoprenoid quinone content of H. pylori, H. cinaedi, and H. fennelliae has been determined and identifies menaquinone-6 (2-methyl-3-farnesyl-farnesyl-1,4-naphthoquinone) and a second, unidentified quinone as major respiratory quinones (51).

Methods for the Differentiation of Helicobacter Species

Classical Phenotypic Characteristics

Most routine laboratories apply the same basic biochemical tests for the identification and differentiation of all Campylobacter-like organisms and would fail to identify many Helicobacter species. Although the number of Helicobacter species encountered in human clinical samples is fairly small, the lack of application of highly standardized procedures and the well-known biochemical inertness of Campylobacter-like organisms render biochemical identification of all of these bacteria very difficult. Whereas Arcobacter strains can be differentiated from Campylobacter and Helicobacter strains by their ability to grow in air and at low temperature (79), there are no clear biochemical characteristics to separate the genus Helicobacter from the genus Campylobacter. Theoretically, one has to differentiate over 35 validly named species and subspecies, as well as various unnamed taxa.

An overview of biochemical and other methods to differentiate Campylobacter and Arcobacter species was described earlier (75). A summary of the characteristics of cultivated Helicobacter species shows that discrimination between some species may rely on only one differential feature (Table 1). Moreover, some species, notably H. pylori and H. acinonychis, and H. felis and H. bizzozeronii, cannot be differentiated with conventional phenotypic tests. In addition, it should be noted that H. pullorum bears a close resemblance to certain Campylobacter species (notably C. lari).

DNA-DNA Hybridization

In taxonomic practice, the reference method for the delineation and identification of bacterial species is determination of the level of DNA-DNA hybridization (81). Strains are considered to belong to a single species if their whole genome DNA-DNA hybridization level is about 70% or greater. The fastidious growth characteristics of many Helicobacter species hamper the isolation of sufficient quantities of highly purified high molecular weight DNA required for these hybridization experiments. Yet, a number of DNA-DNA hybridization studies have been performed within and between Helicobacter species. Significant but low levels of DNA-DNA hybridization (below 70%) have been reported between (i) H. pylori and H. mustelae (21); (ii) H. cinaedi, H. fennelliae, and H. canis (68); and (iii) H. felis, H. bizzozeronii, and H. salomonis (38). Most other species have not been included in quantitative DNA-DNA hybridization studies.

Protein Electrophoresis

It is not practical to implement DNA-DNA hybridizations in a routine laboratory or to use it for routine identification in a reference laboratory. The comparison of whole-cell protein patterns obtained by highly standardized sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) has proven to be extremely reliable to screen and identify large numbers of strains. Numerous studies revealed a correlation between high similarity in whole-cell protein content and level of DNA-DNA hybridization (78). Like for Campylobacter species, PAGE of whole-cell proteins has also been a most successful method for the differentiation of helicobacters (7, 40, 67, 76, 77, 79, 80), and strains of all Helicobacter species are readily distinguished by means of simple numerical analysis. However, this method is not appropriate for routine identification studies because it is laborious, time-consuming, and technically demanding to run the patterns in a sufficiently standardized way.

Cellular Fatty Acid Analysis

The total cellular fatty acid methyl ester composition is a stable parameter provided that highly standardized culture conditions are used. Comparison of fatty acid profiles is of little value if different culture conditions or extraction procedures are used. However, it is a simple, inexpensive, and rapid method that is highly automated. Only a few of the presently known Helicobacter species have been included in published studies (27, 41). H. cinaedi, H. fennelliae, H. pylori, H. mustelae, and H. nemestrinae were readily distinguished (27, 41).

DNA Base Ratio

The DNA G + C ratio of Helicobacter species generally ranges from 30 to 48 mol%. Table 1 lists the known DNA base compositions of the cultivated Helicobacter species.

Ribosomal DNA Restriction Analysis

Restriction profile analysis of PCR amplicons derived from the 23S rRNA gene (34) was shown to differentiate 13 Helicobacter species (including "F. rappini") but cannot unequivocally discriminate H. felis, H. bizzozeronii, and H. salomonis (37).

DNA Probes and PCR Assays

Specific oligonucleotide probes or PCR assays have been described for H. pylori (54) and several other Helicobacter species, including H. pametensis, the Helicobacter Bird-B and Bird-C groups, H. felis, H. hepaticus, H. pullorum, H. bilis, H. trogontum, H. canis, "Candidatus Helicobacter suis," and "Candidatus Helicobacter bovis" (3, 810, 19, 25, 48, 68). It should be stressed that, because of the constant developments in the taxonomy of Helicobacter species, none of these probes or PCR assays have been fully evaluated against all species presently described. For instance, the 16S rDNA gene sequences of multiple strains of H. felis, H. bizzozeronii, and H. salomonis have become available during the past few years, revealing a 98 to 100% similarity in 16S rDNA sequence among strains of these species. The specificity of the H. felis PCR assay (25) should be reevaluated in light of these new findings.

Sequence Analysis of the Small Subunit rDNA

Comparison of nearly complete 16S rDNA sequences is probably the most powerful tool to establish the phylogenetic neighborhood of an unknown organism. Sequence analysis of 16S RNA genes has become a popular tool and is accessible to a wide scientific community. Particularly with organisms that are otherwise difficult to identify, such as helicobacters, it has become common practice to determine the 16S rDNA sequence, or a fragment of it, and compare this with sequences available in public databases. The pitfalls of such an identification approach will be discussed below.

Validated and Candidate Helicobacter Species

Detailed discussion of the gastric Helicobacter species other than H. pylori and of the enterohepatic Helicobacter species appears elsewhere in this volume (chapters 43 and 44) and in a recent review (66). Here we identify each of these species (Tables 2 and 3), group them phylogenetically by 16S rRNA sequence (Table 4, Fig. 1), and highlight specific taxonomic issues where appropriate.

Table 2. Gastric Helicobacter taxa.

Table 2

Gastric Helicobacter taxa.

Table 3. Enterohepatic Helicobacter taxa.

Table 3

Enterohepatic Helicobacter taxa.

Table 4. Distance matrix based on 16S rRNA comparisons.

Table 4

Distance matrix based on 16S rRNA comparisons.

Figure 1. Phylogenetic tree for 18 validated Helicobacter species, 2 Candidatus species, and 9 additional provisional species, based on 16S rRNA sequence.

Figure 1

Phylogenetic tree for 18 validated Helicobacter species, 2 Candidatus species, and 9 additional provisional species, based on 16S rRNA sequence. The scale bar represents a 1% difference in nucleotide sequence as determined by measuring the length of horizontal (more...)

Gastric Helicobacter Species

The well-documented host range of gastric Helicobacter species extends from humans and nonhuman primates to a variety of domesticated and feral land mammals. However, it is likely that the extent of gastric Helicobacter species is even broader than this. A preliminary report (22) described a Helicobacter species cultured from the true (glandular) stomach of the white-sided dolphin (Lagenorhynchus acutus). There is also microscopic evidence that reptiles have gastric spiral bacteria, which will likely be one or more Helicobacter species (Karen Terio, personal communication). The defining feature of gastric Helicobacter species is that they all express urease, which appears to be essential for colonization of this niche. Other biochemical and morphologic characteristics are variable (Table 1).

There are currently seven validly named gastric Helicobacter species, two Candidatus species, and one species that has been described but not yet validated (Table 2). Uncultivated bacteria referred to descriptively as gastrospirillum or as "Gastrospirillum hominis" were identified as helicobacters when 16S rRNA analysis showed that they clearly belong to the Helicobacter genus (65). The bacterium was tentatively designated "H. heilmannii," after Konrad Heilmann, a German histopathologist who at the time had described the largest series of cases and who died prematurely shortly after its publication. However, 16S rRNA sequences between clones derived from two patients differed significantly, suggesting that they might be different species. We originally referred to these as "H. heilmannii" type 1 and "H. heilmannii" type 2 (65). "Candidatus Helicobacter suis" is nearly identical by 16S rRNA gene sequence to "H. heilmannii" type 1, whereas "H. heilmannii" type 2 clusters with H. felis, H. salomonis, H. bizzozeronii, and others. The taxonomic difficulties raised by these and other morphologically indistinguishable bacteria are discussed below in more detail.

H. nemestrinae, isolated from the pigtailed macaque (Macaca nemestrina), was reported to differ from H. pylori by virtue of its growth at 42°C and cellular fatty acid profile (5). Studies of DNA-DNA hybridization and 16S rDNA also suggested that it was a novel species most closely related to H. pylori. Surprisingly, the G + C content was only 24%, much lower than that of all known Helicobacter species, which prompted a reappraisal. The results suggest that H. nemestrinae (ATCC 49396) may in fact be identical to H. pylori. Repeat determination of the 16S rDNA sequence (Floyd Dewhirst, unpublished observations) showed that it is 99.5% identical to human H. pylori and 100% identical to H. pylori isolated from the rhesus macaque (13). Repeat analysis of DNA base composition indicates that H. nemestrinae (ATCC 49396) is 39% G + C (identical to H. pylori) and that the protein profile is also consistent with H. pylori (Peter Vandamme, unpublished observations). Confirmation must await publication of these results.

Enterohepatic Helicobacter Species

The first enterohepatic Helicobacter species was isolated from conventional Wistar rats and BALB/c mice by Phillips and Lee in 1983 (58), almost 10 years before it would be formally named H. muridarum. Since then, a diverse group of Helicobacter species has been found in the intestinal tract and/or hepatobiliary system of humans, other mammals, and birds. Because of potential implications for a broad range of research with laboratory rodents, mice and rats have been most intensively studied, though the host range of these organisms is clearly broad and probably not yet fully defined.

There are currently 11 validated enterohepatic Helicobacter species and several others that await validation or further description (Table 3). Organisms isolated from feces of terns, gulls, a house sparrow, and swine were identified as three biotypes of Helicobacter, originally denoted Helicobacter sp. Bird-A, -B, and -C. The Helicobacter Bird-A isolated was later classified as H. pametensis (10); the other closely related organisms are not formally named and are still referred to as Helicobacter sp. Bird-B and Bird-C because they are represented by only two isolates for one species and one for the other (10). Similarly, Helicobacter CLO-3 isolated from a rectal culture in a homosexual man (16) appears to represent a unique species (68) but has not been formally named because additional isolates have not been identified.

Unlike helicobacters that colonize the stomach, expression of urease in enterohepatic Helicobacter species is variable (Table 1). While these organisms are not normally found in the stomach, occasionally urease-positive species can exploit an altered gastric microenvironment and displace the gastric spirals. This phenomenon has been observed primarily with H. muridarum (42). Some enterohepatic Helicobacter species superficially resemble Campylobacter species but are longer and have a single polar flagellum at each end. Others have periplasmic fibers that wrap helically around the body of the bacterium and give a cross-hatched appearance in negative stain electron micrographs. This morphologic distinction is descriptively convenient, but it has no phylogenetic basis. Like the large group of organisms with the gastrospirillum morphology, which are phylogenetically diverse, recent data suggest that strains that have been called "Flexispira rappini" represent multiple Helicobacter taxa, including the two named species, H. bilis and H. trogontum.

Identification of Novel Helicobacter Species

The Problem of Intraspecies Divergence of rDNA Genes

Like for other fastidious and inert organisms, comparison of partial or nearly complete 16S rDNA sequences has regularly been used to identify novel Helicobacter isolates. Although unsurpassed in its capacity to reveal the phylogenetic neighborhood of an unknown bacterium, comparison of entire 16S rDNA sequences and certainly of partial sequences is not always adequate for species-level identification of strains. There is a lack of knowledge not only of the strain-to-strain variation within a species, but also of the interoperon variation within a single strain. An important pitfall is that 16S rDNA sequences may be too conserved to reveal diversity among species. In Helicobacter species, this is illustrated by comparison of 16S rDNA gene sequences of multiple H. felis, H. bizzozeronii, and H. salomonis strains (38). Although DNA-DNA hybridization studies demonstrated unambiguously that these three taxa represent three distinct species (all three can be distinguished by whole-cell protein electrophoresis as well), all three species have virtually identical 16S rDNA genes (38) (Fig. 1). This failure of 16S rDNA sequence analysis to distinguish closely related species is obviously not restricted to the Helicobacter genus (17, 78).

The lack of sufficient variability in 16S rDNA sequences is not the only rDNA-related problem. Helicobacter isolates have been classified as novel species or putative novel species based on a distinct position in a phylogenetic tree, and on one or a few differential biochemical characteristics, thereby ignoring the putative intraspecies diversity in the 16S rDNA gene sequence or in the biochemical reactivity pattern. The reported intraspecies diversity in 16S rDNA sequences of some Campylobacter and Helicobacter species is exceptionally high (31, 77). Whereas intraspecies diversity in 16S rDNA sequence of up to 3% is generally not unusual, differences up to 4.3% among C. hyointestinalis strains (31) and up to 4.5% among H. cinaedi strains (77) have been reported. Some of these H. cinaedi strains share higher percentages of their 16S rDNA sequence with strains of species like H. canis, H. bilis, or H. fennelliae than with other strains of H. cinaedi. In a phylogenetic tree based on 16S rDNA, such strains may cluster far apart and erroneously suggest that they represent novel species (35, 77).

In such a situation when no additional taxonomic analyses are performed, it is important to realize that there are no criteria to balance the significance of detected phenotypic differences among isolates. "H. westmeadii" strains, although grouping very closely with H. cinaedi reference strains in the 16S rDNA phylogenetic tree, were considered to be distinct species because of their anaerobic growth requirements and supposed biochemical differences from H. cinaedi reference strains (74). The failure to grow the isolates under optimal conditions (a microaerobic atmosphere with hydrogen) probably led to at least a few equivocal biochemical test results. When grown under optimal conditions and compared with the biochemical profile of a large panel of H. cinaedi strains, the "H. westmeadii" isolates conformed to the general biochemical reactivity pattern of H. cinaedi (77). It is essential to use an independent taxonomic tool to balance the significance of such biochemical differences when they occur. In the case of "H. westmeadii," both isolates were readily identified as H. cinaedi by means of whole-cell fatty and whole-cell protein analysis (74, 77), indicating that the biochemical differences revealed intraspecies diversity rather than interspecies diversity.

The value of 16S rDNA sequence analysis as a tool to reveal the taxonomic neighborhood of unclassified isolates is beyond dispute. However, whole-cell protein or fatty acid analysis, extended biochemical testing, or restriction profile analysis of PCR amplicons derived from the 23S rRNA gene (see above) should be considered to endorse tentative identification results obtained by comparison of complete 16S rDNA genes, and to balance the significance of potential biochemical differences. As useful as the 16S rDNA sequence analysis method is, present data clearly indicate that it cannot be regarded as the gold standard for species-level identification of helicobacters and other epsilon Proteobacteria species. It should be clear that further descriptions of putative new species should be made with due care and attention to the present view that interstrain relationships should be described by means of a polyphasic taxonomic analysis.

The Problem of Morphologically Defined Taxa: "Flexispira" and "Gastrospirillum"

"Flexispira rappini"

"Flexispira rappini" is a provisional name given to gram-negative, microaerobic, motile, spindle-shaped bacteria with spiral periplasmic fibers and bipolar tufts of sheathed flagella. Strains with this distinctive morphology have been isolated from various sources including aborted lambs, canine feces and gastric mucosa, intestinal mucosa of laboratory mice, and pig intestines (2, 40, 62) and from stool and blood samples of children and adults (67, 71). Strains described as "Flexispira rappini" belong to the genus Helicobacter by phylogenetic analysis and share their unusual morphological features with several named Helicobacter species, including H. bilis (19) and H. trogontum (48). Considerable diversity in the 16S rRNA gene of 35 strains exhibiting these distinctive morphological characteristics has been observed (11). A polyphasic investigation of the relationships between all these strains is required to clarify the taxonomic position and status of this complex group within the genus Helicobacter. However, at present the name "Flexispira rappini" refers to a characteristic morphotype shared by several distinct taxa and not to a single well-defined species. Dewhirst et al. have suggested that these organisms be referred to as Helicobacter sp. flexispira, with a specific taxon designation when appropriate (11), until further characterization is completed.

"Gastrospirillum"

Nomenclatural and taxonomic problems also exist for the group of gastric bacteria often referred to as "Gastrospirillum" spp. or "H. heilmannii" (below referred to as "gastrospirilla"). These organisms are principally characterized by their cell morphology (large, tightly coiled rods without periplasmic fibers), and such cells have been observed in gastric biopsies of many hosts, including humans, cats, dogs, pigs, monkeys, rats, and various captive exotic carnivores (36, 40, 47, 49). Gastrospirilla are also characterized by their extremely fastidious growth requirements, and much of the uncertainty concerning their taxonomic status has arisen since most investigators have failed to culture the strains in vitro. Phylogenetic analyses clearly identify all gastrospirilla as helicobacters. In pigs, a provisional name, "Candidatus Helicobacter suis" has been proposed. However, the 16S rDNA sequences of these porcine bacteria are indistinguishable from those of gastrospirilla observed in some humans (16S rDNA sequence "type 1" human gastrospirilla) and in several nonhuman primates (53, 65). In humans, a second type (as defined by its 16S rDNA sequence) has been observed as well. These "type 2" human gastrospirilla have a 16S rDNA sequence that is indistinguishable from that of H. felis, H. bizzozeronii, and H. salomonis, and the first cultured human isolate was identified as H. bizzozeronii (39). Both human types of gastrospirilla are commonly referred to as "Gastrospirillum hominis" and as "H. heilmannii," but the names "H. heilmannii" type 1 and type 2 are at present most commonly used in medical microbiology. In clinical gastroenterology, where only morphologic data are available, these organisms are referred to collectively as "H. heilmannii."

The H. felis–bizzozeronii–salomonis phylogenetic lineage now comprises three validly named species. There are no molecular diagnostic tools that permit distinction among these three species, including 16S rDNA sequence, which lacks discriminatory power. Therefore, as long as the large majority of these "H. heilmannii" type 2 bacteria remain uncultured, it is not possible to determine to which of the presently known species they belong or to determine if they possibly represent one or more additional species. The same is true for the organisms belonging to the "H. heilmannii" type 1–"Candidatus Helicobacter suis" phylogenetic lineage.

Since all gastrospirilla are Helicobacter species, the terms "Gastrospirillum" and "Gastrospirillum hominis" should be abandoned, though "gastrospirilla" may be used descriptively. The designation "Candidatus Helicobacter suis" was proposed based on the identification in swine of gastrospirilla that had a 16S rRNA gene that was nearly identical to that from "H. heilmannii" type 1. However, organisms with virtually identical 16S rRNA sequences have also been commonly found in rhesus monkeys and other nonhuman primates, as well as in additional human patients (50, 53). If these organisms all represent the same species (recall that the H. felis–bizzozeronii–salomonis phylogenetic lineage is equally homogeneous and comprises at least three distinct species), then its host range would appear broad and the epithet "suis" may be misleading. At this stage the objective data for decision making are not available, and formal nomenclatural proposals should be published in appropriate taxonomic journals. However, the present confusion is too important to be ignored, and it may take several years before the true taxonomic relationships between porcine and human gastrospirilla are determined. It is timely to attempt to clarify part of this confusion by proposing a common terminology for the different groups of gastrospirilla. Whenever 16S rRNA sequence data are available, it should be specified if the organisms belong to the H. felis–bizzozeronii–salomonis phylogenetic lineage or to the "H. heilmannii" type 1–"Candidatus Helicobacter suis" phylogenetic lineage. Members of the former could be referred to as the H. felis-like gastrospirilla, which would apply to what we have previously called "H. heilmannii" type 2 (65). Members of the latter group of gastrospirilla may be referred to as "H. heilmannii"-like gastrospirilla, which would apply to what we have previously called "H. heilmannii" type 1 (65). The designation "H. heilmannii"-like is justified by the fact that it is in common use, appropriately pays tribute to an early worker in the field, and avoids the implication that the organism has a restricted host range. Ideally, a single term, different from the others, should be used for all these organisms when phylogenetic information is not available. The terms "gastrospirilla" or "human gastrospirilla" are descriptive terms that would obviously be appropriate. Using the term "H. heilmannii-like" descriptively, in the absence of genetic information, may obscure species differences that will be apparent when 16S rRNA sequence or better cultivation methods are available.

H. pylori is Panmictic but Exists as a Single Species

The 16S rRNA gene is useful as a molecular clock to establish phylogenetic relationships because it is both highly constrained and sufficiently divergent to permit differentiation of species in most cases. Examination of 16S rRNA genes from multiple H. pylori strains shows virtually no heterogeneity (15, 33). On the other hand, there is extensive genetic heterogeneity among strains of H. pylori that is attributable largely to horizontal gene transfer and free recombination (70). Even when compared to bacterial species that have a known nonclonal population structure, such as Neisseria meningitidis, H. pylori stands alone in the extent to which there is allelic diversity (chapter 32). Some have therefore suggested that H. pylori may exist as a species complex (32). As additional DNA sequence data accumulate, this genetic heterogeneity may become even more apparent. Whether H. pylori represents multiple species should be determined with current internationally accepted criteria for bacterial speciation. At present, despite its panmictic population structure, a polyphasic approach to taxonomy that is based on level of whole-genome DNA-DNA hybridization clearly identifies H. pylori as a single species.

Conclusions

There are currently 18 formally validated Helicobacter species. In addition, two candidate species have been described, "Candidatus Helicobacter bovis" and "Candidatus Helicobacter suis." The intense interest in these organisms ensures that the Helicobacter genus will continue to grow at a brisk pace, as currently described species are validated and novel species are discovered. To avoid the confusion that has sometimes accompanied premature formal naming of species, the International Committee of Systematic Bacteriology Subcommittee on the Taxonomy of Campylobacter and Related Bacteria has agreed on minimum requirements for the description of new species of the genus Helicobacter (12). This is a polyphasic approach that is based on examination of five or more strains, with analysis of phenotypic characteristics, an essentially complete (greater than 1,450 bases) 16S rRNA sequence, and DNA-DNA hybridization. Numerical analysis of whole-cell protein profiles may be considered as an alternative or adjunctive method to DNA-DNA hybridization. Putative new species of uncultured organisms for which 16S rRNA data are available may be assigned to Candidatus status if five or more putative isolates are identified, and if sequence data are supported by in situ hybridization. Adherence to these guidelines will simplify the taxonomy of the Helicobacter genus, and in so doing, will ultimately contribute to our understanding of the role of diverse Helicobacter species in the pathogenesis of gastric and enterohepatic diseases in humans and other animals.

Acknowledgment

Work in the laboratory of JVS is supported by grant A142081 from the National Institutes of Health.

References

1.
Anonymous Validation of the publication of new names and new combinations previously effectively published outside the IJSB: list no. 17. Int. J. Syst. Bacteriol. 1985;35:223–225. [PubMed: 9776615]
2.
Archer J. R., Romero S., Ritchie A. E., Hamacher M. E., Steiner B. M., Bryner J. H., Schell R. F. Characterization of an unclassified microaerophilic bacterium associated with gastroenteritis. J. Clin. Microbiol. 1988;26:101–105. [PMC free article: PMC266203] [PubMed: 3343302]
3.
Battles J. K., Williamson J. C., Pike K. M., Gorelick P. L., Ward J. M., Gonda M. A. Diagnostic assay for Helicobacter hepaticus based on nucleotide sequence of its 16S rRNA gene. J. Clin. Microbiol. 1995;33:1344–1347. [PMC free article: PMC228160] [PubMed: 7542270]
4.
Bode G., Mauch F., Ditschuneit H., Malfertheiner P. Identification of structures containing polyphosphate in Helicobacter pylori. J. Gen. Microbiol. 1993;139:3029–3033. [PubMed: 8126429]
5.
Brondson M., Goodwin C., Sly L., Chilvers T., Schoenknecht F. Helicobacter nemestrinae sp. nov., a spiral bacterium found in the stomach of a pigtailed macaque (Macaca nemestrina) Int. J. Syst. Bacteriol. 1991;41:148–153. [PubMed: 1995031]
6.
Chalk P. A., Roberts A. D., Blows W. M. Metabolism of pyruvate and glucose by intact cells of Helicobacter pylori studied by 13C NMR spectroscopy. Microbiology. 1994;140:2085–2092. [PubMed: 7921258]
7.
Costas M., On S. L. W., Owen R. J., Lopez-Urquijo B., Lastovica A. J. Differentiation of Helicobacter species by numerical analysis of their one-dimensional electrophoretic protein patterns. Syst. Appl. Microbiol. 1993;16:396–404.
8.
De Groote D., van Doorn L.-J., Ducatelle R., Verschuuren A., Haesebrouck F., Quint W. G. V., Jalava K., Vandamme P. `Candidatus Helicobacter suis,' a gastric Helicobacter from pigs, and its phylogenetic relatedness to other gastrospirilla. Int. J. Syst. Bacteriol. 1999;49:1769–1777. [PubMed: 10555359]
9.
De Groote D., van Doorn L. J., Ducatelle R., Verschuuren A., Tilmant K., Quint W. G. V., Haesebrouck F., Vandamme P. Phylogenetic characterization of `Candidatus Helicobacter bovis', a new gastric helicobacter in cattle. Int. J. Syst. Bacteriol. 1999;49:1707–1715. [PubMed: 10555352]
10.
Dewhirst F., Seymour C., Fraser G., Paster B. J., Fox J. Phylogeny of Helicobacter isolates from bird and swine feces and description of Helicobacter pametensis sp. nov. Int. J. Syst. Bacteriol. 1994;44:553–560. [PubMed: 7520743]
11.
Dewhirst F. E., Fox J. G., Mendes E. N., Paster B. J., Gates C. E., Kirkbride C. A., Eaton K. A. Flexispira rappini" strains represent at least ten Helicobacter taxa. Int. J. Syst. Evol. Microbiol. 2000;50:1781–1787. [PubMed: 11034487]
12.
Dewhirst F. E., Fox J. G., On S. L. W. Proposal of minimal standards for describing new species of the genus Helicobacter. Int. J. Syst. Evol. Microbiol. 2000;50:2231–2237. [PubMed: 11156001]
13.
Drazek E. S., Dubois A., Holmes R. K. Characterization and presumptive identification of Helicobacter pylori isolates from rhesus monkeys. J. Clin. Microbiol. 1994;32:1799–1804. [PMC free article: PMC263799] [PubMed: 7523441]
14.
Eaton K., Dewhirst F., Radin M. Helicobacter acinonyx sp. nov., isolated from cheetahs with gastritis. Int. J. Syst. Bacteriol. 1993;43:99–106. [PubMed: 8379970]
15.
Eckloff B. W., Poszorski R. P., Kline B. C., Cockerill F. R. A comparison of 16S ribosomal DNA sequences from five isolates of Helicobacter pylori. Int. J. Syst. Bacteriol. 1994;44:320–323. [PubMed: 8186097]
16.
Fennell C. L., Totten P. A., Quinn T. C., Patton D. L., Holmes K. K., Stamm W. E. Characterization of Campylobacter-like organisms isolated from homosexual men. J. Infect. Dis. 1984;149:58–66. [PubMed: 6693790]
17.
Fox G. E., Wisotzkey J. D., Jurtshuk, Jr P. How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. Int. J. Syst. Bacteriol. 1992;42:166–170. [PubMed: 1371061]
18.
Fox J., Dewhirst F., Tully J., Paster B., Yan L., Taylor N., Collins M., Gorelick P., Ward J. Helicobacter hepaticus sp. nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice. J. Clin. Microbiol. 1994;32:1238–1245. [PMC free article: PMC263656] [PubMed: 8051250]
19.
Fox J., Yan L., Dewhirst F., Paster B. J., Shames B., Murphy J., Hayward A., Beicher J., Mendes E. Helicobacter bilis sp. nov., a novel Helicobacter species isolated from bile, livers, and intestines of aged, inbred mice. J. Clin. Microbiol. 1995;33:445–454. [PMC free article: PMC227964] [PubMed: 7536217]
20.
Fox J. G., Chien C. C., Dewhirst F. E., Paster B. J., Shen Z., Melito P. L., Woodward D. L., Rodgers F. G. Helicobacter canadensis sp. nov. isolated from humans with diarrhea: an example of an emerging pathogen. J. Clin. Microbiol. 2000;38:2546–2549. [PMC free article: PMC86964] [PubMed: 10878041]
21.
Fox J. G., Chilvers T., Goodwin C. S., Taylor N. S., Edmonds P., Sly L. J., Brenner D. Campylobacter mustelae, a new species resulting from the elevation of Campylobacter pylori subsp. mustelae to species status. Int. J. Syst. Bacteriol. 1989;39:301–303.
22.
Fox J. G., Harper C. M., Xu S., Dewhirst F. E., Paster B. J. Isolation and characterization of urease positive novel Helicobacter spp. isolated from gastric mucosa of white sided dolphins (Lagenorhynchus acutus) Gastroenterology. 2000;118:A2568. [PMC free article: PMC92375] [PubMed: 11055919]
23.
Franklin C., Beckwith C., Livingston R., Riley L., Gibson S., Besch-Williford C., Hook R. J. Isolation of a novel Helicobacter species, Helicobacter cholecystus sp. nov., from the gallbladders of syrian hamsters with cholangiofibrosis and centrilobular pancreatitis. J. Clin. Microbiol. 1996;34:2952–2958. [PMC free article: PMC229440] [PubMed: 8940429]
24.
Franklin C. L., Riley L. K., Livingston R. S., Beckwith C. S., Hook, Jr R. R., Besch-Williford C. L., Hunziker R., Gorelick P. L. Enteric lesions in SCID mice infected with "Helicobacter typhlonicus," a novel urease-negative Helicobacter species. Lab. Anim. Sci. 1999;49:496–505. [PubMed: 10551450]
25.
Germani Y., Dauga C., Duval P., Huerre M., Levy M., Pialoux G., Sansonetti P., Grimont P. A. Strategy for the detection of Helicobacter species by amplification of 16S rRNA genes and identification of H. felis in a human gastric biopsy. Res. Microbiol. 1997;148:315–326. [PubMed: 9765810]
26.
Goodwin C. S., Armstrong J. A., Chilvers T., Peters M., Collins M. D., Sly L., McConnel W., Harper W. E. S. Transfer of Campylobacter pylori and Campylobacter mustelae to Helicobacter gen. nov. as Helicobacter pylori comb. nov. and Helicobacter mustelae comb. nov., respectively. Int. J. Syst. Bacteriol. 1989;39:397–405.
27.
Goodwin C. S., McConnell W., McCulloch R. K., McCullough C., Hill R., Bronsdon M. A., Kasper G. Cellular fatty acid composition of Campylobacter pylori from primates and ferrets compared with those of other campylobacters. J. Clin. Microbiol. 1989;27:938–943. [PMC free article: PMC267458] [PubMed: 2745703]
28.
Goodwin C. S., McCulloch R. K., Armstrong J. A., Wee S. H. Unusual cellular fatty acids and distinctive ultrastructure in a new spiral bacterium (Campylobacter pyloridis) from the human gastric mucosa. J. Med. Microbiol. 1985;19:257–267. [PubMed: 3981612]
29.
Goto K., Ohashi H., Ebukuro S., Itoh K., Tohma Y., Takakura A., Wakana S., Ito M., Itob T. Isolation and characterization of Helicobacter species from the stomach of the house musk shrew (Suncus murinus) with chronic gastritis. Curr. Microbiol. 1998;37:44–51. [PubMed: 9625789]
30.
Hänninen M.-L., Happonen I., Saari S., Jalava K. Culture and characteristics of Helicobacter bizzozeronii, a new canine gastric Helicobacter sp. Int. J. Syst. Bacteriol. 1996;46:160–166. [PubMed: 8573490]
31.
Harrington C. S., On S. L. Extensive 16S rRNA gene sequence diversity in Campylobacter hyointestinalis strains: taxonomic and applied implications. Int. J. Syst. Bacteriol. 1999;49(Pt 3):1171–1175. [PubMed: 10425776]
32.
Hazell S. L., Andrews R. H., Mitchell H. M., Daskalopoulous G. Genetic relationship among isolates of Helicobacter pylori: evidence for the existence of a Helicobacter pylori species-complex. FEMS Microbiol. Lett. 1997;150:27–32. [PubMed: 9163902]
33.
Hook-Nikanne J., Solin M.-L., Kosunen T. U., Kaartinen M. Comparison of partial 16S rRNA sequences of different Helicobacter pylori strains, Helicobacter mustelae and a gastric Campylobacter-like organism (GCLO) Syst. Appl. Microbiol. 1991;14:270–274.
34.
Hurtado A., Owen R. J. A rapid identification scheme for Helicobacter pylori and other species of Helicobacter based on 23S rRNA gene polymorphisms. Syst. Appl. Microbiol. 1997;20:222–231.
35.
Husmann M., Gries C., Jehnichen P., Woelfel T., Gerken G., Ludwig W., Bhakdi S. Helicobacter sp. strain Mainz isolated from an AIDS patient with septic arthritis: case report and nonradioactive analysis of 16S rRNA sequence. J. Clin. Microbiol. 1994;32:3037–3039. [PMC free article: PMC264221] [PubMed: 7883895]
36.
Jakob W., Stolte M., Valentin A., Schröder H. D. Demonstration of Helicobacter pylori-like organisms in the gastric mucosa of captive exotic carnivores. J. Comp. Pathol. 1997;116:21–33. [PubMed: 9076597]
37.
Jalava K., Hielm S., Hirvi U., Hänninen M. L. Evaluation of a molecular identification scheme based on 23S rRNA gene polymorphisms for differentiating canine and feline gastric Helicobacter spp. Lett. Appl. Microbiol. 1999;28:269–274. [PubMed: 10212438]
38.
Jalava K., Kaartinen M., Utriainen M., Happonen I., Hänninen M. L. Helicobacter salomonis sp. nov., a canine gastric Helicobacter sp. related to Helicobacter felis and Helicobacter bizzozeronii. Int. J. Syst. Bacteriol. 1997;47:975–982. [PubMed: 9336895]
39.
Jalava, K., S. L. W. On, C. S. Harrington, L. P. Andersen, M.-L. Hänninen, and P. A. R. Vandamme. 1999. "Helicobacter heilmannii," a human gastric pathogen, and H. bizzozeronii, a frequent canine gastric coloniser, represent the same species, abstr. HD5, p. 116. In 10th International Workshop on Campylobacter, Helicobacter, and Related Organisms. Baltimore, Md.
40.
Jalava K., On S. L. W., Vandamme P. A., Happonen I., Sukura A., Hänninen M. L. Isolation and identification of Helicobacter spp. from canine and feline gastric mucosa. Appl. Environ. Microbiol. 1998;64:3998–4006. [PMC free article: PMC106591] [PubMed: 9758832]
41.
Lambert M. A., Patton C. M., Barrett T. J., Moss C. W. Differentiation of Campylobacter and Campylobacter-like organisms by cellular fatty acid composition. J. Clin. Microbiol. 1987;25:706–713. [PMC free article: PMC266064] [PubMed: 3571478]
42.
Lee A., Chen M., Coltro N., O'Rourke J., Hazell S., Hu P., Li Y. Long term infection of the gastric mucosa with Helicobacter species does induce atrophic gastritis in an animal model of Helicobacter pylori infection. Zentralbl. Bakteriol. 1993;280:38–50. [PubMed: 8280955]
43.
Lee A., Phillips M. W., O'Rourke J. L., Paster B. J., Dewhirst F. E., Fraser G. J., Fox J. G., Sly L. I., Romaniuk P. J., Trust T. J., Kouprach S. Helicobacter muridarum sp. nov., a microaerophilic helical bacterium with a novel ultrastructure isolated from the intestinal musosa of rodents. Int. J. Syst. Bacteriol. 1992;42:27–36. [PubMed: 1736969]
44.
Linton D., Clewley J. P., Burnens A., Owen R. J., Stanley J. An intervening sequence (IVS) in the 16S rRNA gene of the eubacterium Helicobacter canis. Nucleic Acids Res. 1994;22:1954–1958. [PMC free article: PMC308106] [PubMed: 7518076]
45.
Marshall B. J., Goodwin C. S. Revised nomenclature of C. pyloridis. Int. J. Syst. Bacteriol. 1987;37:68.
46.
Marshall B. J., Warren J. R. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet. 1984;i:1311–1315. [PubMed: 6145023]
47.
McNulty C. A., Dent J. C., Curry A., Uff J. S., Ford G. A., Gear M. W., Wilkinson S. P. New spiral bacterium in gastric mucosa. J. Clin. Pathol. 1989;42:585–591. [PMC free article: PMC1141985] [PubMed: 2738164]
48.
Mendes E., Queiroz D., Dewhirst F., Paster B., Moura S., Fox J. Helicobacter trogontum sp. nov., isolated from the rat intestine. Int. J. Syst. Bacteriol. 1996;46:916–921. [PubMed: 8863417]
49.
Mendes E. N., Queiroz D. M. M., Rocha G. A., Moura S. B., Leite V. H. R., Fonseca M. E. F. Ultrastructure of a spiral micro-organism from pig gastric mucosa ("Gastrospirillum suis") J. Med. Microbiol. 1990;33:61–66. [PubMed: 2231673]
50.
Morgner A., Lehn N., Andersen L. P., Thiede C., Bennedsen M., Trebesius K., Neubauer B., Neubauer A., Stolte M., Bayerdörffer E. Helicobacter heilmannii-associated primary gastric low-grade MALT lymphoma: complete remission after curing the infection. Gastroenterology. 2000;118:821–882. [PubMed: 10784580]
51.
Moss C. W., Lambert-Fair M. A., Nicholson M. A., Guerrant G. O. Isoprenoid quinones of Campylobacter cryaerophila, C. cinaedi, C. fennelliae, C. hyointestinalis, C. pylori, and "C. upsaliensis. J. Clin. Microbiol. 1990;28:395–397. [PMC free article: PMC269621] [PubMed: 2312688]
52.
Murray R. G., Stackebrandt E. Taxonomic note: implementation of the provisional status Candidatus for incompletely described procaryotes. Int. J. Syst. Bacteriol. 1995;45:186–187. [PubMed: 7857801]
53.
O'Rourke, J. L., B. Neilan, and A. Lee. 1999. Phylogenetic relationship of Helicobacter heilmannii-like organisms originating from humans and animals, abstr. HE4, p. 118. In 10th International Workshop on Campylobacter, Helicobacter, and Related Organisms, Baltimore, Md.
54.
On S. L. Identification methods for campylobacters, helicobacters, and related organisms. Clin. Microbiol. Rev. 1996;9:405–422. [PMC free article: PMC172901] [PubMed: 8809468]
54a.
Page R. D. M. TREEVIEW: an application to display phylogenetic trees on personal computers. Comput. Applic. Biosci. 1996;12:357–358. [PubMed: 8902363]
55.
Paster B. J., Lee A., Fox J. G., Dewhirst F. E., Tordoff L. A., Fraser G. J., O'Rourke J. L., Taylor N. S., Ferrero R. Phylogeny of Helicobacter felis sp. nov., Helicobacter mustelae, and related bacteria. Int. J. Syst. Bacteriol. 1991;41:31–38. [PubMed: 1704791]
56.
Pearson A. D., Bamforth J., Booth L., Holdstock G., Ireland A., Walker C., Hawtin P., Millward-Sadler H. Polyacrylamide gel electrophoresis of spiral bacteria from the gastric antrum. Lancet. 1984;ii:1349–1350. [PubMed: 6145041]
57.
Pearson, A. D., and Great Britain Public Health Laboratory Service. 1983. Campylobacter II: Proceedings of the Second International Workshop on Campylobacter Infections, Brussels, 6–9 September 1983. Public Health Laboratory Service, London, United Kingdom.
58.
Phillips M. W., Lee A. Isolation and characterization of a spiral bacterium from the crypts of rodent gastrointestinal tracts. Appl. Environ. Microbiol. 1983;45:675–683. [PMC free article: PMC242343] [PubMed: 6402981]
59.
Robertson, B. R., J. L. O'Rourke, P. Vandamme, S. L. W. On, and A. Lee. Helicobacter ganmani sp. nov., an anaerobic, urease negative Helicobacter species isolated from the intestines of laboratory mice. Submitted for publication. [PubMed: 11594622]
60.
Romaniuk P. J., Zoltowska B., Trust T. J., Lane D. J., Olsen G. J., Pace N. R., Stahl D. A. Campylobacter pylori, the spiral bacterium associated with human gastritis, is not a true Campylobacter sp. J. Bacteriol. 1987;169:2137–2141. [PMC free article: PMC212113] [PubMed: 3571163]
61.
Saunders K. E., Shen Z., Dewhirst F. E., Paster B. J., Dangler C. A., Fox J. G. Novel intestinal Helicobacter species isolated from cotton-top tamarins (Saguinus oedipus) with chronic colitis. J. Clin. Microbiol. 1999;37:146–151. [PMC free article: PMC84191] [PubMed: 9854080]
62.
Schauer D. B., Ghori N., Falkow S. Isolation and characterization of "Flexispira rappini" from laboratory mice. J. Clin. Microbiol. 1993;31:2709–2714. [PMC free article: PMC265986] [PubMed: 7504685]
63.
Shen Z., Fox J., Dewhirst F., Paster B., Foltz C., Van L., Shames B., Perry L. Helicobacter rodentium sp. nov., a urease negative Helicobacter species isolated from laboratory mice. Int. J. Syst. Bacteriol. 1997;47:627–634. [PubMed: 9226892]
64.
Simmons J. H., Riley L. K., Besch-Williford C. L., Franklin C. L. Helicobacter mesocricetorum sp. nov., a novel Helicobacter isolated from the feces of syrian hamsters. J. Clin. Microbiol. 2000;38:1811–1817. [PMC free article: PMC86596] [PubMed: 10790105]
65.
Solnick J. V., O'Rourke J., Lee A., Paster B., Dewhirst F. E., Tompkins L. S. An uncultured gastric spiral organism is a newly identified Helicobacter in humans. J. Infect. Dis. 1993;168:379–385. [PubMed: 8335974]
66.
Solnick J. V., Schauer D. B. Emergence of diverse Helicobacter species in the pathogenesis of gastric and enterohepatic diseases. Clin. Microbiol. Rev. 2001;14:59–97. [PMC free article: PMC88962] [PubMed: 11148003]
67.
Sorlin P., Vandamme P., Nortier J., Hoste B., Rossi C., Pavlof S., Struelens M. J. Recurrent "Flexispira rappini" bacteremia in an adult patient undergoing hemodialysis: case report. J. Clin. Microbiol. 1999;37:1319–1323. [PMC free article: PMC84762] [PubMed: 10203478]
68.
Stanley J., Linton D., Burnens A., Dewhirst F., Owen R., Porter A., On S., Costas M. Helicobacter canis sp. nov., a new species from dogs: an integrated study of phenotype and genotype. J. Gen. Microbiol. 1993;139:2495–2504. [PubMed: 8254320]
69.
Stanley J., Linton D., Burnens A., Dewhirst F., Porter A., Owen R., Costas M. Helicobacter pullorum, sp. nov.—genotype and phenotype of a new species isolated from poultry and from human patients with gastroenteritis. Microbiology. 1994;140:3441–3449. [PubMed: 7533595]
70.
Suerbaum S., Smith J. M., Bapumia K., Morelli G., Smith N. H., Kunstmann E., Dyrek I., Achtman M. Free recombination within Helicobacter pylori. Proc. Natl. Acad. Sci. USA. 1998;95:12619–12624. [PMC free article: PMC22880] [PubMed: 9770535]
71.
Tee W., Leder K., Karroum E., Dyall-Smith M. Flexispira rappini" bacteremia in a child with pneumonia. J. Clin. Microbiol. 1998;36:1679–1682. [PMC free article: PMC104899] [PubMed: 9620399]
72.
Tomb J.-F., White O., Kerlavage A. R., Clayton R. A., Sutton G. G., Fleischmann R. D., Ketchum K. A., Klenk H. P., Gill S., Dougherty B. A., Nelson K., Quackenbush J., Zhou L., Kirkness E. F., Peterson S., Loftus B., Richardson D., Dodson R., Khalak H. G., Glodek A., McKenney K., Fitzegerald L. M., Lee N., Adams M. D., Hickey E. K., Berg D. E., Gocayne J. D., Utterback T. R., Peterson J. D., Kelley J. M., Cotton M. D., Weidman J. M., Fujii C., Bowman C., Watthey L., Wallin E., Hayes W. S., Borodovsky M., Karp P. D., Smith H. O., Fraser C. M., Venter J. C. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature. 1997;388:539–547. [PubMed: 9252185]
73.
Totten P. A., Fennell C. L., Tenover F. C., Wezenberg J. M., Perine P. L., Stamm W. E., Holmes K. K. Campylobacter cinaedi (sp. nov.) and Campylobacter fennelliae (sp. nov.): two new Campylobacter species associated with enteric disease in homosexual men. J. Infect. Dis. 1985;151:131–139. [PubMed: 3965584]
74.
Trivett-Moore N. L., Rawlinson W. D., Yuen M., Gilbert G. L. Helicobacter westmeadii sp. nov., a new species isolated from blood cultures of two AIDS patients. J. Clin. Microbiol. 1997;35:1144–1150. [PMC free article: PMC232719] [PubMed: 9114397]
75.
Vandamme, P. 2000. Taxonomy of the family Campylobacteriaceae, p. 3–26. In M. Blaser (ed.), Campylobacter. American Society for Microbiology, Washington, D.C.
76.
Vandamme P., Falsen E., Pot B., Kersters K., De Ley J. Identification of Campylobacter cinaedi isolated from blood and feces of children and adult females. J. Clin. Microbiol. 1990;28:1016–1020. [PMC free article: PMC267856] [PubMed: 2191002]
77.
Vandamme P., Harrington C. S., Jalava K., On S. L. W. Misidentifying helicobacters: the Helicobacter cinaedi example. J. Clin. Microbiol. 2000;38:2261–2266. [PMC free article: PMC86777] [PubMed: 10834986]
78.
Vandamme P., Pot B., Gillis M., de Vos P., Kersters K., Swings J. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev. 1996;60:407–438. [PMC free article: PMC239450] [PubMed: 8801440]
79.
Vandamme P., Pot B., Kersters K. Differentiation of campylobacters and Campylobacter-like organisms by numerical analysis of one-dimensional electrophoretic protein patterns. Syst. Appl. Microbiol. 1991;14:57–66.
80.
van der Ven A. J., Kullberg B. J., Vandamme P., Meis J. F. Helicobacter cinaedi bacteremia associated with localized pain but not with cellulitis. Clin. Infect. Dis. 1996;22:710–711. [PubMed: 8729212]
81.
Wayne L. G., Brenner D. J., Colwell R. R., Grimont P. A. D., Kandler P., Krichevsky M. I., Moore L. H., Moore W. E. C., Murray R. G. E., Stackebrandt E., Starr M. P., Trüper H. G. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int. J. Syst. Bacteriol. 1987;37:463–464.