Taxonomy of Campylobacter, Arcobacter, Helicobacter and related bacteria: current status, future prospects and immediate concerns

Summary, 1S 2. Introduction, 1S 3. Taxonomy of Campylobacter: a brief history, 2S 4. Current taxonomic status of ‘campylobacteria’, 2S 4.1 Campylobacter and Bacteroides ureolyticus, 2S 4.2 Arcobacter, 5S 4.3 Sulfurospirillum and ‘Dehalospirillum’, 6S 4.4 Helicobacter, ‘Gastrospirillum’ and ‘Flexispira’, 6S 4.5 Wolinella, Thiovulum and Thiomicrospira, 7S 4.6 Other taxa, 7S 5. Species diversity and misclassification: immediate problems for applied microbiologists, 8S 5.1 Identification of bacteria: a brief introduction, 8S 5.2 Phenotypic variation, 8S 5.3 Ribosomal RNA gene sequence variation by intervening sequence elements, 8S 5.4 Sequence variation by genetic drift, 9S 5.5 Ribosomal RNA gene sequence similarity between taxa, 9S 5.6 The dangers of misclassification, 10S 5.7 Misidentification: the human element, 10S 6. Future prospects, 10S 7. Acknowledgements, 12S 8. References, 12S The taxonomy of the genus Campylobacter has changed dramatically since its inception in 1963. At that time the genus comprised just two species. At present, taxa that were once assigned to Campylobacter may belong to one of over 50 species distributed among six genera. Most of these taxa belong to a phylogenetically distinct group referred to as either ribosomal RNA (rRNA) superfamily VI or the epsilon division of the class Proteobacteria. The taxonomic diversity of the group is matched by the diverse habitats in which they may be found, and by the wide range of diseases that they are associated with. Recognition of their clinical and economic importance has resulted in intense interest in the group, and the application of increasingly sophisticated isolation, detection and chemotaxonomic methods continues to elucidate new aspects of their biodiversity. However, despite the advances in new bacterial systematics, there remain a number of important issues concerning the classification of various campylobacterial taxa that require careful consideration. Ultimately, these issues are relevant to many working in the field of applied microbiology, including clinicians, veterinarians, epidemiologists and taxonomists. The purpose of this article is briefly to review the major developments in the taxonomy of Campylobacter from its inception to the present day; summarize the most recent changes in the field; analyse current topical issues of special relevance to applied microbiologists, including identification of the bacteria; and speculate on future prospects for campylobacterial taxonomy. The science of taxonomy comprises three principal areas: classification, identification and nomenclature, each linked to the other. Strains are ordered, or classified, into groups on the basis of some common feature(s), and traits allowing the group to be identified (i.e. discriminated from similar taxa) are defined. For practical purposes, the taxon must be named and, for the sake of clarity, the name must be formed with reference to rules of nomenclature. Taxonomy aims to provide a meaningful biological framework upon which a wide range of other sciences is built. However, the inaccurate delineation of taxa serves to confound workers in a variety of disciplines. The genus Campylobacter was formed in 1963 and its taxonomic structure has changed extensively since its inception. The use of increasingly sophisticated molecular methods has revealed fascinating aspects of campylobacterial biodiversity. Nonetheless, certain proposals have proven controversial, and several important problems require investigation. The aim of this paper is to provide a brief overview of the current status of the taxonomy of Campylobacter and related bacteria; analyse current topical issues of special relevance to applied microbiologists; and speculate on future developments in the field. Readers seeking more detailed appraisals of taxonomic developments concerning these taxa from the 1800s to the early 1990s are directed elsewhere (61). The genus Campylobacter was first proposed in 1963 by 45 and included just two species, Campylobacter fetus and ‘Campylobacter bubulus’ (now Campylobacter sputorum; see section 4.1). These taxa were formerly classified as Vibrio spp. until Sebald and Véron applied Hugh and Leifson’s test for fermentative metabolism, and DNA base composition, to distinguish them from ‘true’Vibrio spp. However, most of the scientific community continued to refer to these taxa as ‘Vibrio fetus’ and ‘Vibrio bubulus’, until a more extensive investigation of the genus was made (66). This study used serological and biochemical analyses, and DNA base composition, to study relationships between ‘V. fetus’, ‘V. bubulus’ and several other misclassified vibrios described by various workers but not included in the original generic proposal, namely ‘Vibrio jejuni’, ‘Vibrio coli’, ‘Vibrio sputorum’ and ‘Vibrio faecalis’. Although the taxonomic ranks of ‘C. bubulus’, C. sputorum and ‘Campylobacter faecalis’ would subsequently change or be refined (see section 4.1), Campylobacter was established as a distinct, recognizable genus. By coincidence, the study of 4 greatly increased interest in Campylobacter by indicating their high prevalence in human diarrhoea. Since C. fetus was already recognized as an important animal pathogen as a cause of abortion and infectious infertility, the identification of Campylobacter as a cause of enteric disease in humans ensured the attention of both clinicians and veterinarians alike. As a consequence, and aided by a greatly improved understanding in growth characteristics and isolation methods, 12 new species or subspecies were discovered in a range of different diseases and habitats from 1974 to 1988 (reviewed by 61). The taxonomic status of many of these taxa would change (42; 16; 65; 37) owing to the wider application of advanced taxonomic methods. Indeed, the potential of the 16S rRNA gene for determining phylogenetic relationships among all living organisms had attracted much interest (69) and would play a major role in an extensive rearrangement of Campylobacter taxonomy. Numerical comparison of partial 16S rRNA gene sequences identified several distinct clades within Campylobacter (41). Of these, ‘Campylobacter pylori’ and ‘Campylobacter mustelae’ (from human and ferret gastric mucosa respectively) were reclassified into a new genus, Helicobacter, to reconcile major differences from other Campylobacter spp. in flagellar structure, fatty acid and menaquinone composition, and 16S rRNA gene sequences (16). 41 also identified ‘Campylobacter cryaerophila’ and ‘Campylobacter nitrofigilis’ as related, yet distinct from other Campylobacter and Helicobacter spp., and several Wolinella and Bacteroides spp. appeared to be affiliated to either Campylobacter (‘Wolinella curva’, ‘Wolinella recta’, ‘Bacteroides gracilis’, Bacteroides ureolyticus) or Helicobacter (Wolinella succinogenes). Even Thiovulum, an uncultivable marine bacterium, appeared to be distantly related to these taxa. These issues were resolved in an extensive polyphasic taxonomic study of the entire Campylobacter complex (60). By use of DNA–rRNA hybridizations, relative phylogenetic positions were determined and data cross-referenced with other phenotypic and genetic data. This study provided the basis of the taxonomic structure used at present, and delineated Campylobacter spp. as a diverse yet phylogenetically distinct group, rRNA superfamily VI (also known as the ɛ-division of the Proteobacteria;54). This comprised rRNA homology groups I (Campylobacter and B. ureolyticus), II (Arcobacter) and III (Helicobacter and W. succinogenes). The close relatedness of rRNA homology groups I and II was noted and a subsequent paper proposed these be included in the family Campylobacteraceae (58). 60 also examined two saprophytic, sulphur-reducing strains described as ‘Campylobacter-like’ or ‘Spirillum sp.’ that appeared to be related, yet separate, from the other rRNA homology groups of rRNA superfamily VI. These strains would later form a new genus, Sulphurospirillum (44). At present, the genus Campylobacter contains 16 species and six subspecies (Fig. 1). Campylobacter jejuni subsp. jejuni, C. jejuni subsp. doylei, Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis and Campylobacter helveticus form a genetically close group of species which (C. jejuni subsp. doylei aside) are the most commonly isolated from human and animal diarrhoea. ‘Campylobacter hyoilei’, isolated from lesions of porcine proliferative enteritis, was later identified by a wide range of phenotypic and genotypic methods as a strain of C. coli (64). It was, however, suggested that these strains may represent a pathogenic variant (pathovar) of C. coli and the identification of a genetic marker that is evidently specific to ‘C. hyoilei’, and the observation of some metabolic differences (6), makes this prospect feasible. Dendrogram showing relationships between 62 strains of Campylobacter, Arcobacter, Helicobacter, Wolinella, Thiovulum, Bacteroides ureolyticus, and as by comparison of 16S rRNA gene sequences and The sequence were and a and were not in the The species Campylobacter Campylobacter Campylobacter Campylobacter Campylobacter C. sputorum and C. to be related Most in the human C. has in the human and C. sputorum is also in the enteric and of various Campylobacter sputorum comprises three by on the of a strain to or sputorum in both and Strains described as were reclassified as since the these two taxa to and were not Campylobacter was to be uncultivable methods, and was first C. in of its taxonomic status This species has by use of an to bacterial from Campylobacter by most similar to C. sputorum with which a similar and a common Campylobacter fetus subsp. C. fetus subsp. Campylobacter subsp. and C. subsp. are also similar by and Campylobacter fetus is in C. subsp. is enteric in and C. subsp. in the Campylobacter diversity at the 16S rRNA genetic a with for Campylobacter from workers as later in this number of taxonomic problems in Campylobacter Campylobacter was first isolated from the but there have several of strains associated with with a to that of C. jejuni However, some strains from as as relatedness to the strain in By current strains to a species relatedness to each other the C. strain and these represent genetically distinct species, yet there are other phenotypic or genotypic to distinguish and C. is as a The of C. in both and has used to that its in is or at However, at present be determined all the strains represent a species distinct but related the C. is a to this and methods for and of C. this the of potential Campylobacter is also known to be distinct are namely the a variant and a and the two groups are related to C. but to there has to the taxonomic status of these strains from were to be by and and DNA several distinct groups The taxonomic status of these different and groups in of the wide of these recent study that and strains form related but distinct in a of a The subspecies division of C. fetus a special for of C. fetus subsp. fetus cause of abortion in and from C. fetus subsp. cause of infectious is since the disease be is The subspecies is within the genus in that is on these different pathogenic Although several phenotypic and genotypic methods are for the two subspecies of these to the strain classification and identification of C. fetus subspecies be the between and of the diversity in the 16S rRNA gene for some taxa (see section has for the taxonomic status of a new species, C. which with strains of the C. subspecies (Fig. Indeed, C. subsp. 16S rRNA gene sequence of with C. in this At this of similarity see section 16S rRNA sequence to taxa represent the or distinct, species and are to the taxonomic of However, between the two taxa have not yet and the of C. is in with of the habitats of C. be to C. is of species or a subspecies an of C. Dendrogram showing relationships between Campylobacter C. related Campylobacter spp. and Bacteroides ureolyticus, as by comparison of 16S rRNA gene sequences and The sequence were and a and were not in the The taxonomic of the B. of 16S rRNA gene sequences this species as an to the Campylobacter group see and several phenotypic differences between B. and Campylobacter spp. have including fatty acid and Nonetheless, the of this species is to that of many as by a of phenotypic traits of campylobacterial strains However, the prospect of B. to the genus Campylobacter be on its diversity are as suggested the genus comprised in animal and human enteric and from reclassified Campylobacter spp. of growth and at (60). species have to the genus since its inception. was first described as a Campylobacter but an extensive polyphasic taxonomic study of organisms assigned ‘Campylobacter to Arcobacter, on the basis of DNA–rRNA was described in the study and comprised strains from various by and that two of be on the basis of fatty acid and It is that these subspecies but the of biochemical to them has a contains species at present (Fig. 1). However, of bacterial of and a have revealed the of organisms that 16S rRNA gene sequence similarity with taxa, species. Although 60 described the taxonomic of two ‘Campylobacter-like’ strains and as distinct within rRNA superfamily they were not classified until the growth DNA base composition, and DNA of with C. sputorum and W. As a of the differences the genus Sulfurospirillum was proposed to both and comprised the strain the taxonomic status of at this study of sulphur-reducing revealed 16S rRNA sequences of the two strains formed a distinct within rRNA superfamily VI and classified two other strains into Of these, one was named which was to have a fatty acid and menaquinone for of rRNA superfamily VI. The other strain named was later named by also described by a from 16S rRNA sequence identified one other Sulfurospirillum strain but this was not classified by The distinct phylogenetic positions of and that these represent species. was from and has the to a This and its distinct in a phylogenetic were for its as a of a new genus and species to rRNA superfamily VI. However, other Sulfurospirillum spp. were not included for of 16S rRNA gene sequences in the Sulfurospirillum (Fig. and the many of of and DNA base with Sulfurospirillum spp. These data that to the genus The intense interest in as a cause of human related including gastric has many to a variety of for similar The that some species Helicobacter and Helicobacter were associated with also to the wide attention to the genus. continues to be by the potential of some species Helicobacter Helicobacter Helicobacter Helicobacter and indicating an with (reviewed by As a consequence, the number of Helicobacter spp. described (i.e. in the of and formerly the of since has increased dramatically from to as of an division and taxa are gastric or enteric in a enteric species the certain and are most associated with The gastric species were isolated from of various and and Helicobacter Helicobacter Helicobacter and Helicobacter The enteric species Helicobacter Helicobacter Helicobacter Helicobacter Helicobacter and Helicobacter by as yet taxa have proposed as or from and from by several species have named in other and have in at These the later in this later in this and by to the a range of as yet taxa have described in various that are by their in phylogenetic to represent Helicobacter spp. are several taxonomic issues to be resolved in number of taxa are recognized by their as and have referred to as the generic name first suggested for strains in some human organisms have in a wide range of other including and various with and suggested for in and (see 16S rRNA gene sequence all known as Helicobacter spp. have proven to in and this has greatly taxonomic to the among strains in various However, a have from and and three distinct species are and strains a species, The human strains to as either or are into two distinct sequence strains form a with strains are affiliated to and and in the 16S rRNA gene of these species is that be The taxonomic status of the human strains in to the complex and to has to However, a polyphasic taxonomic has identified a human strain as This not that all human are the 16S rRNA gene sequence of is more similar to that of (Fig. 1). data the two sequence of human represent at two distinct, species. This to the potential of these The of polyphasic taxonomic on require advances in methods. The name was used to from and human and later The of this is the is by a complex of and has of Although several strains this to Helicobacter is from certain that all strains belong to a species. 16S sequence of several Helicobacter spp. that included two strains of human and porcine delineated them to two different clades both and are distinct species these the of described strains from that were and different from a human The of the 16S rRNA gene at the species (see 5.4 and to strains from each of the described strains of a sequence represent distinct or a species. is a for chemotaxonomic to relationships among all strains the Since the of Wolinella by changes have made and W. a of the the species. 16S rRNA gene sequence have Thiovulum to be affiliated to rRNA superfamily VI the is not in to the of the genus contains one species, Thiovulum and is marine in The genus contains species, of which six are to rRNA superfamily VI. However, is since 16S rRNA gene sequence is affiliated to Thiovulum and rRNA superfamily VI (Fig. 1). The of is a by Thiovulum and some spp., but is to its taxonomic within the taxa that were first classified as Campylobacter spp. have subsequently to represent species in that are phylogenetically distinct from the entire rRNA superfamily VI. as the of porcine proliferative was first named ‘Campylobacter 16S rRNA sequence the to be more related to several have identified similar in various other including and group of from and first described as ‘C. have named phylogenetic and The genus two species, and is since of these of enteric and are associated with and acid from is in spp. and 16S rRNA sequence to the of the For most applied microbiologists, the identification of a bacterial strain their most common with taxonomy. identification of a strain is to clinical prevalence of a species, or as a to the and of various Identification the comparison of data for an strain with of known taxa. Strains are identified two data with to an The used for may represent from biochemical test to or DNA sequences as by the of in or by 16S rRNA gene The of a on of the of diversity that may be for a in a of species diversity in strain and, strains are subsequently as taxa, the potential for in future in the of a species is a established of potential for the known in this is that of C. jejuni strains the to the test for this species from C. Other C. sputorum and C. fetus subsp. Other methods, as fatty acid and similar problems in certain groups provided a of for the of molecular that were to be more and to identification on biochemical This was not Since the a new to has described gene are with and of strains with of known taxa) and data genetic diversity have These are to the of many genetic methods was the use of gene sequences 16S as both a for similarity and also as a basis for the of DNA and in the rRNA gene be and the of identification on which is sequences have in 16S of all C. sputorum and and some C. C. C. C. and strains The rRNA of all C. fetus and some strains of C. coli, C. jejuni, C. C. C. Helicobacter and may also sequences may in and base composition, and are not in of the rRNA sequences problems for and into a in a between the may the of the molecular is a or this or the of several C. jejuni subsp. jejuni reference strains at the a test to be specific for this species and from the rRNA gene different are in some both in the Although 12 recognized the of on the the of and with to was not recognized at that from reference Campylobacter jejuni subsp. jejuni strains examined with a on rRNA gene sequence data are a of intervening sequence in the rRNA molecular marker VI Since identification are from 16S or rRNA sequence and require the of a much of these they to be more to the contains a recognized by one of the in the the more The must be that a strain a may not represent a but an one in which an is of the of on a identification for Helicobacter is by sequence not represent the by which rRNA may some species, the of sequence variation between strains be extensive and not to a specific of the At present, C. and are known to extensive variation in their 16S rRNA that as as have described between strains of the species These have that two strains 16S rRNA gene sequence similarity are to represent distinct require the of ɛ-division at current data that a similarity distinct species with more the C. most strains of each subspecies into distinct Sequence not the of with data in but also identification methods on sequence data. in a has the potential to cause a noted that in the 16S rRNA gene of C. had the potential to of the of a identification for this species for and C. jejuni, on and in the gene of methods be that Ribosomal RNA gene sequence between related species also a It is established that the taxonomic of the 16S rRNA gene be for related species For strains of and sequence similarity in their 16S rRNA despite in and Other groups of taxa known to similarity in their 16S rRNA and and C. coli, C. jejuni and C. Campylobacter and Campylobacter C. C. and C. and and and the two C. fetus subspecies this is some to that some strains of C. coli and C. jejuni 16S sequences identification of several species be by of the 16S sequence of an strain to the species is of from taxa. of to sequence data not data and (i.e. sequence and (i.e. similar problems for some species the rRNA gene of and diversity to strain misclassification, which in in strain ‘C. hyoilei’, strain and ‘Campylobacter were all to be species, by of their in a phylogenetic and three of subsequent comparison of a phenotypic to the taxa (reviewed by polyphasic taxonomic established methods have each of the taxa belong to species. and strain are ‘C. is C. coli and ‘Campylobacter is C. subsp. The resulted from an of the diversity in species and the use of test phenotypic and methods. Nonetheless, data biochemical test for other workers to strains for as or strain Indeed, there is at one strain in data that a wide range and and is Future prevalence may have and strain had not recognized as also by of data by The sequence for strain was the original had from a different This may the by of a strain that to the original of 2. the is that a the of a polyphasic to identification and classification is that may be data are and methods with are For complex groups as Campylobacter and related this is However, for be a strain is identified as a taxon that has not by in the It is certain that new taxa to be to Campylobacter its related genera. The of these in and other continues to many to their role and prevalence in as as has that are one to The is to that with isolation or detection methods. Although several groups have proven to be C. isolation methods have with and attention Although and be made to these methods, these for the and are also to as a of new taxa. taxa Sulfurospirillum and have in a variety of and and have the to a variety of as and as These be in and this is to bacterial to the diversity and application of in different The of 16S rRNA gene sequence on campylobacterial taxonomy to be Although this for related species, is at present for phylogenetic with 16S rRNA gene sequence data from of and belong to ɛ-division (Fig. the of campylobacterial diversity to be from 16S rRNA sequences of strains of the and (Campylobacter fetus subsp. Sulfurospirillum Helicobacter Wolinella of the Proteobacteria. The of as yet from and is within were by the the The sequence Species identification continues to be a The of new methods for this purpose is The of the 16S rRNA gene for close relationships workers to other as or as new for molecular The use of several in sequence provide new into phylogenetic of genotypic also It is to distinguish the related species C. and C. fetus to subspecies by of was the has similar potential but owing to the use of two and in of The of in molecular would in to by and has for of 16 Campylobacter spp. and a comparison of discriminated all also with other and data Dendrogram of the on in of Campylobacter fetus and C. strains the The on the the as determined by the and were formed at the similarity Taxonomy is a and Most of the recent advances in understanding campylobacterial have from advances in taxonomic methods and from bacterial It is that in in in campylobacterial classification, the polyphasic to bacterial is to remain a in which methods play an important role It is also important to that the present methods for phylogenetic relationships are not and that of bacterial have methods and may have on campylobacterial is certain that the field to be as and as has since 1963. The and of for strains used in this study many and for the 16S rRNA sequence of Thiovulum used in this