Campylobacter Cryaerophila Research Articles

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

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.

Natural and Experimental Infections of Campylobacter cryaerophila in Rainbow Trout. Gross Pathology, Bacteriology, Clinical Pathology and Chemotherapy.

Natural and Experimental Infections of Campylobacter cryaerophila in Rainbow Trout. Gross Pathology, Bacteriology, Clinical Pathology and Chemotherapy.

Arcobacter cryaerophilus and Arcobacter butzleri isolated from preputial fluid of boars and fattening pigs in Brazil.

Figure 1. Restriction fragment length polymorphism (RFLP) patterns of representative Arcobacter isolates from male pigs. Reference strains of A. butzleri and A. cryaerophilus groups 1A and 1B are compared with field isolates. All of the field isolates hybridized with the probe and thus were verified as Arcobacter. The arrow indicates the position of the ;3.0-kb band that is characteristic of A. butzleri. Isolate SP51 exhibited this ;3.0-kb band and was thus verified as A. butzleri. On the basis of RFLP patterns, the remaining isolates could be identified as A. cryaerophilus 1A (SP32, SP35, and SP56) and 1B (SP31, SP34, SP57). Hind III digest of bacteriophage l is the size standard (kb). Aerotolerant Campylobacter-like organisms were first isolated from aborted bovine and porcine fetuses in the 1970s.2,3 These organisms were designated a single species, Campylobacter cryaerophila, on the basis of aerotolerance and the ability to grow at 25 C.9 In 1991, after extensive DNA homology studies, the species Campylobacter butzleri was proposed for the aerotolerant Campylobacter-like organisms isolated from human enteritis.7 On the basis of that study, two distinct groups of Campylobacter cryaerophila, 1A and 1B, were identified. In 1992, the genus Arcobacter was proposed to encompass the aerotolerant microorganisms of 4 species: Arcobacter nitrofigilis, A. cryaerophilus, A. butzleri, and A. skirrowii.14,16 The species of Arcobacter associated with human or animal disease are Arcobacter, A. cryaerophilus (groups 1A and 1B), and A. skirrowii. Of these, A. butzleri is isolated most frequently from human infections. Arcobacter butzleri has been isolated from water, poultry, and ground pork, all of which pose a risk for human infection.1,6,12,18 Previous studies have detected Arcobacter in ground pork samples obtained from midwest slaughter facilities, with recoveries from the 5 plants ranging from 0 to 90%.1 Whether management practices at the source farms or the sanitary conditions during slaughter influenced the prevalence of Arcobacter spp. in ground pork was not determined. Aerotolerant Campylobacter-like organisms have been isolated from aborted and normal porcine fetuses, sows with reproductive problems, and clinically healthy specific-pathogen-free pigs.2–5,9,10,17,18,20 In one study, 90% of Campylobacter spp. isolated from fetuses and 78% of Campylobacter spp. isolated from porcine vaginal swabs were identified as C. cryaerophila. Strains from reproductively impaired and

Arcobacter-specific and Arcobacter butzleri-specific 16S rRNA-based DNA probes

The genus Arcobacter encompasses gram-negative, aerotolerant, spiral-shaped bacteria formerly designated Campylobacter cryaerophila. Two genus-specific 16S rRNA-based oligonucleotide DNA probes (23-mer and 27-mer) were developed. The probes hybridized with strains of Arcobacter butzleri (n = 58), Arcobacter cryaerophilus (n = 19), and Arcobacter skirrowii (n = 17). The probes did not cross-react with any of the reference strains of Campylobacter, Helicobacter, including "Flexispira rappini," or Wolinella. The 27-mer hybridized with 61 Arcobacter spp. field isolates originating from late-term aborted porcine (n = 54) and equine (n = 2) fetuses and humans with enteritis (n = 5). The species of Arcobacter isolates (n = 56) recovered from aborted livestock fetuses were determined by ribotyping and were as follows: A. cryaerophilus group 1A (11 of 56; 20%), A. cryaerophilus group 1B (37 of 56; 66%), A. butzleri (5 of 56; 9%), and unknown (3 of 56; 5%). The five human field strains were identified as A. butzleri. A species-specific DNA probe (24-mer) for A. butzleri was also developed since there is evidence that this organism may be a human pathogen. This probe hybridized with previously characterized strains of A. butzleri (n = 58), with 10 field strains identified as A. butzleri by ribotyping and with 2 strains having an indeterminate ribotype. The A. butzleri-specific probe did not cross-react with strains of A. skirrowii (n = 17) and A. cryaerophilus (n = 19).

Specific detection of Campylobacter jejuni and Campylobacter coli by using polymerase chain reaction.

Development of a routine detection assay for Campylobacter jejuni and Campylobacter coli in clinical specimens was undertaken by using the polymerase chain reaction (PCR). An oligonucleotide primer pair from a conserved 5' region of the flaA gene of C. coli VC167 was used to amplify a 450-bp region by PCR. The primer pair specifically detected 4 strains of C. coli and 47 strains of C. jejuni; but it did not detect strains of Campylobacter fetus, Campylobacter lari, Campylobacter upsaliensis, Campylobacter cryaerophila, Campylobacter butzleri, Campylobacter hyointestinalis, Wolinella recta, Helicobacter pylori, Escherichia coli, Shigella spp., Salmonella spp., Vibrio cholerae, Citrobacter freundii, or Aeromonas spp. By using a nonradioactively labeled probe internal to the PCR product, the assay could detect as little as 0.0062 pg of purified C. coli DNA, or the equivalent of four bacteria. In stools seeded with C. coli cells, the probe could detect between 30 and 60 bacteria per PCR assay. The assay was also successfully used to detect C. coli in rectal swab specimens from experimentally infected rabbits and C. jejuni in human stool samples.

Polyphasic taxonomic study of the emended genus Arcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., an aerotolerant bacterium isolated from veterinary specimens.

The relationships of 77 aerotolerant Arcobacter strains that were originally identified as Campylobacter cryaerophila (now Arcobacter cryaerophilus [P. Vandamme, E. Falsen, R. Rossau, B. Hoste, P. Segers, R. Tytgat, and J. De Ley, Int. J. Syst. Bacteriol. 41:88-103, 1991]) and 6 reference strains belonging to the taxa Arcobacter nitrofigilis, Arcobacter cryaerophilus, and "Campylobacter butzleri" were studied by using a polyphasic approach, in which we performed DNA-rRNA hybridizations, DNA-DNA hybridizations, a numerical analysis of whole-cell protein patterns after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an analysis of cellular fatty acid compositions, and a phenotypic analysis and determined DNA base ratios. Our results indicate that "C. butzleri" should be transferred to the genus Arcobacter as Arcobacter butzleri comb. nov., as was suggested by Kiehlbauch and coworkers (J. A. Kiehlbauch, D. J. Brenner, M. A. Nicholson, C. N. Baker, C. M. Patton, A. G. Steigerwalt, and I. K. Wachsmuth, J. Clin. Microbiol. 29:376-385, 1991). A rapid screening of all strains in which we used the sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique revealed five major groups, which were identified by using DNA-DNA hybridization data as A. cryaerophilus (two distinct electrophoretic subgroups), A. butzleri, A. nitrofigilis, and a new species, for which we propose the name Arcobacter skirrowii. The phylogenetic position within rRNA superfamily VI was established for each species. A. butzleri strains and strains belonging to one of the electrophoretic subgroups of A. cryaerophilus had similar fatty acid contents. An analysis of fatty acid compositions allowed clear-cut differentiation of all of the other groups. All of the species could be distinguished by using classical phenotypic tests, although erroneous identifications due to a shortage of clear-cut differentiating tests could occur.

Restriction fragment length polymorphisms in the ribosomal genes for species identification and subtyping of aerotolerant Campylobacter species

Whole-cell chromosomal digests of 84 strains of aerotolerant Campylobacter (AC) were examined by using PvuII restriction fragment length polymorphisms of rRNA genes followed by hybridization with Escherichia coli 16S and 23S rRNA (ribotyping). The AC strains belonged to Campylobacter cryaerophila (n = 13) and a newly defined species, "C. butzleri" (n = 64). Strains of C. cryaerophila belonged to two hybridization groups: DNA group 1A (including the type strain of C. cryaerophila) and DNA group 1B (J. A. Kiehlbauch, D. J. Brenner, M. A. Nicholson, C. N. Baker, C. M. Patton, A. G. Steigerwalt, and I. K. Wachsmuth, J. Clin. Microbiol. 29:376-385, 1991). Six AC strains not classified as C. cryaerophila or "C. butzleri" were also included. All 35 sporadic human and animal isolates of "C. butzleri" sent to the Centers for Disease Control for identification showed different ribotype patterns. However, most "C. butzleri" strains contained common bands at approximately 3.0, 6.2, 12.0, and 15.0 kb; the 3.0-kb band was present in all but four strains. An additional 23 strains of "C. butzleri," isolated as part of special studies, contained the 3.0-kb band. Thus, on the basis of visual identification of the 3.0-kb band, 94% of available strains were correctly identified as "C. butzleri." Ribotyping demonstrated that C. cryaerophila strains (DNA groups 1A and 1B) were different from C. butzleri strains. All C. cryaerophila strains demonstrated a common ribosomal DNA restriction fragment of 3.2 kb; DNA group 1B strains contained an additional common band at 2.6 kb. Ribotyping patterns of AC species were easily distinguished from patterns of other Campylobacter, Helicobacter, and Wolinella species.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of Group 2 Aerotolerant Campylobacter Species from Thai Children with Diarrhea

Campylobacter species were isolated from 93 (15%) of 631 Thai children with diarrhea using the membrane filter technique on nonselective blood agar incubated at 37 degrees C. Campylobacter jejuni was isolated from 62 (10%), Campylobacter coli from 14 (2%), and atypical campylobacters from 17 (3%). The 17 atypical strains were first characterized biochemically and by dot blot DNA hybridization. Catalase-negative strains also were characterized by DNA hybridization and ribotype pattern. One strain was a catalase-negative "Campylobacter upsaliensis" and another was a nitrate-negative Campylobacter jejuni doylei. Fifteen isolates were aerotolerant strains most closely resembling Campylobacter cryaerophila or "C. upsaliensis" by dot hybridization. These aerotolerant strains, designated group 2 ("Campylobacter butzleri"), had ribotypes distinct from C. cryaerophila and have previously been shown to be related by DNA hybridization at the species level to the group 2 aerotolerant Campylobacter type strain (D2686). Group 2 aerotolerant Campylobacter were the atypical Campylobacter species most frequently isolated from Thai children with diarrhea.

Campylobacter butzleri sp. nov. isolated from humans and animals with diarrheal illness

Seventy-eight aerotolerant Campylobacter isolates were characterized phenotypically and by DNA hybridization (hydroxyapatite method at 50 and 65 degrees C). Two DNA relatedness groups were found. (i) Sixty-four strains belonged to aerotolerant Campylobacter DNA hybridization group 2. These organisms were isolated from humans, primarily with diarrheal illness, and animals on several continents. Strains were aerotolerant at 30 and 36 degrees C and catalase negative or weakly catalase positive, grew in media containing glycine and on MacConkey agar, were susceptible to nalidixic acid, and were resistant to cephalothin. The name Campylobacter butzleri sp. nov. is proposed for this group. (ii) DNA hybridization group 1 consisted of the type strain of Campylobacter cryaerophila and 13 additional strains isolated from 10 animals outside the United States and from three humans within the United States. This group was genetically diverse; five strains were closely related to the type strain of C. cryaerophila (DNA hybridization group 1A), and eight strains were more closely related to one another (DNA hybridization group 1B). Strains in DNA hybridization group 1B were phenotypically diverse, with two of eight strains resembling C. cryaerophila. The seven strains from DNA hybridization groups 1A and 1B which resembled C. cryaerophila and the C. cryaerophila type strain were aerotolerant only at 30 degrees C and catalase positive, did not grow in glycine or on MacConkey agar, were generally susceptible to nalidixic acid, and were resistant to cephalothin. The remaining six strains of DNA hybridization group 1B phenotypically resembled C. butzleri; however, they were generally catalase positive and susceptible to nalidixic acid and cephalothin. DNA hybridization group 1B is not designated as a separate species at this time since it cannot, with certainty, be separated genetically from C. cryaerophila or phenotypically from C. butzleri.

Revision of Campylobacter, Helicobacter, and Wolinella Taxonomy: Emendation of Generic Descriptions and Proposal of Arcobacter gen. nov.

Hybridization experiments were carried out between DNAs from more than 70 strains of Campylobacter spp. and related taxa and either 3H-labeled 23S rRNAs from reference strains belonging to Campylobacter fetus, Campylobacter concisus, Campylobacter sputorum, Campylobacter coli, and Campylobacter nitrofigilis, an unnamed Campylobacter sp. strain, and a Wolinella succinogenes strain or 3H- or 14C-labeled 23S rRNAs from 13 gram-negative reference strains. An immunotyping analysis of 130 antigens versus 34 antisera of campylobacters and related taxa was also performed. We found that all of the named campylobacters and related taxa belong to the same phylogenetic group, which we name rRNA superfamily VI and which is far removed from the gram-negative bacteria allocated to the five rRNA superfamilies sensu De Ley. There is a high degree of heterogeneity within this rRNA superfamily. Organisms belonging to rRNA superfamily VI should be reclassified in several genera. We propose that the emended genus Campylobacter should be limited to Campylobacter fetus, Campylobacter hyointestinalis, Campylobacter concisus, Campylobacter mucosalis, Campylobacter sputorum, Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and "Campylobacter upsaliensis." Wolinella curva and Wolinella recta are transferred to the genus Campylobacter as Campylobacter curvus comb. nov. and Campylobacter rectus comb. nov., respectively. Bacteroides gracilis and Bacteroides ureolyticus are generically misnamed and are closely related to the genus Campylobacter. Campylobacter nitrofigilis, Campylobacter cryaerophila, and an unnamed Campylobacter sp. strain constitute a new genus, for which the name Arcobacter is proposed; this genus contains two species, Arcobacter nitrofigilis comb. nov. (type species) and Arcobacter cryaerophilus comb. nov. Wolinella succinogenes so far is the only species of the genus Wolinella. The genus Helicobacter is also emended; Campylobacter cinaedi and Campylobacter fennelliae are included in this genus as Helicobacter cinaedi comb. nov. and Helicobacter fennelliae comb. nov., respectively. The genus "Flexispira," with "Flexispira rappini" as the only species, is closely related to the genus Helicobacter. The free-living, sulfur-reducing campylobacters do not belong to any of these genera; they probably constitute a distinct genus within rRNA superfamily VI.

Biochemical and serological characterization of Campylobacter cryaerophila

Sixty-two isolates of Campylobacter cryaerophila were recovered from aborted porcine and bovine fetuses, from porcine, bovine, and equine feces, and from different tissues of a dead piglet. Phenotypic characterization was carried out on all isolates, and the results were compared with those obtained with the reference strains of C. cryaerophila, C. jejuni, C. coli, C. laridis, and C. hyointestinalis. The ability of C. cryaerophila strains to grow under aerobic conditions at 16 degrees C was found to be most useful in differentiating them from strains of other Campylobacter species. Studies were undertaken to develop a serotyping system for C. cryaerophila on the basis of the Lior serotyping system for C. jejuni and C. coli by use of a tube agglutination test with formalinized whole-cell (FWC) and boiled whole-cell (BWC) antigens. Antisera against 18 strains of C. cryaerophila were produced in rabbits. Thirty-five percent of C. cryaerophila strains were typed with the FWC suspension as an antigen, and 61% were typed with the BWC suspension as an antigen. None of the C. cryaerophila strains tested autoagglutinated in saline. BWC antigens of C. jejuni, C. coli, and C. laridis cross-reacted with C. cryaerophila, whereas FWC antigens did not cross-react. Neither FWC nor BWC antigens of C. hyointestinalis reacted with C. cryaerophila antisera.

Isoprenoid quinones of Campylobacter cryaerophila, C. cinaedi, C. fennelliae, C. hyointestinalis, C. pylori, and "C. upsaliensis"

The isoprenoid quinone contents of Campylobacter cryaerophila, C. cinaedi, C. fennelliae, C. hyointestinalis, C. pylori, and "C. upsaliensis" were determined by reverse-phase thin-layer and high-performance liquid chromatography. All six of these recently named Campylobacter species contained menaquinone-6 (MK-6), but only C. hyointestinalis and "C. upsaliensis" contained 2,[5 or 8]-dimethyl-3-farnesyl-farnesyl-1,4-naphthoquinone (*MK-6), a previously described novel menaquinone of the Campylobacter genus. C. cryaerophila, C. cinaedi, C. fennelliae, and C. pylori contained an unidentified quinone (Un-MK-6) with a molecular weight of 580 and a base peak ion of m/e = 225 by mass spectrometry but with chromatographic properties different from those of MK-6. *MK-6 and Un-MK-6 are important chemotaxonomic markers of Campylobacter and Campylobacter-like organisms.

Location of double bonds in monounsaturated fatty acids of Campylobacter cryaerophila with dimethyl disulfide derivatives and combined gas chromatography-mass spectrometry.

Location of the double-bond position of monounsaturated fatty acids in Campylobacter cryaerophila was accomplished with combined gas chromatography-mass spectrometry analysis of dimethyl disulfide (DMDS) derivatives. The monoenoic fatty acids from whole bacterial cells were converted to methyl esters and then to DMDS adducts and analyzed by capillary gas chromatography-mass spectrometry. The mass spectra of DMDS adducts gave an easily recognizable molecular ion (M+) and two major diagnostic ions attributable to fragmentation between the two CH3S groups located at the original site of unsaturation. Two previously unidentified acids that distinguish C. cryaerophila from other bacteria were identified as C14:1 omega 7 and C16:1 omega 7 from their mass spectral fragmentation patterns. Resolution of cis and trans isomers by capillary column gas chromatography permitted assignment of the trans isomer to the C16:1 omega 7 acid.

Campylobacter cryaerophila isolated from a human.

Campylobacter cryaerophila was recovered from a single stool specimen of a 35-year-old homosexual man who presented with intermittent diarrhea for 4 to 6 months. The isolate was identified as C. cryaerophila by using biochemical reactions and confirmed by DNA-DNA hybridization and gas-liquid chromatograph profiles. The base composition of DNA of the isolate was found to be 31.1 +/- 1 mol% (G+C). C. cryaerophila was previously reported to be associated with bovine and porcine abortions in animals. We believe this is the first report of recovery of C. cryaerophila from human stool.

Phylogenetic Study of the Genus Campylobacter

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

Taxonomic Position of Campylobacter cryaerophila sp. nov.

A total of 90 aerotolerant Campylobacter strains, which were isolated mainly from animal abortions, were examined by using a wide range of biochemical and physiological tests. The resulting data, together with data for reference Campylobacter strains that were similarly examined, were compared by using several numerical taxonomic analysis methods. Our analyses demonstrated that there is a strong similarity between the aerotolerant strains and strains of Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, and Campylobacter sputorum. For practical purposes this study revealed four possible taxa; one taxon consisted of C. fetus strains, one taxon consisted of the C. sputorum strains, and a third taxon consisted of the thermophilic strains, having C. coli and C. jejuni strains as separate entities. The fourth group consisted of all of the aerotolerant strains and was considered a new species of Campylobacter. All of the aerotolerant strains exhibited a tolerance to lower temperatures and higher oxygen concentrations than previously observed with strains of Campylobacter; the name Campylobacter cryaerophila sp. nov. is suggested for these strains. In this paper we present identification characteristics of C. cryaerophila strains and a description of the new species.