Abstract
Species-specific identification of campylobacters is problematic, primarily due to the absence of suitable biochemical assays and the existence of atypical strains. 16S rRNA gene (16S rDNA)-based identification of bacteria offers a possible alternative when phenotypic tests fail. Therefore, we evaluated the reliability of 16S rDNA sequencing for the species-specific identification of campylobacters. Sequence analyses were performed by using almost 94% of the complete 16S rRNA genes of 135 phenotypically characterized Campylobacter strains, including all known taxa of this genus. It was shown that 16S rDNA analysis enables specific identification of most Campylobacter species. The exception was a lack of discrimination among the taxa Campylobacter jejuni and C. coli and atypical C. lari strains, which shared identical or nearly identical 16S rDNA sequences. Subsequently, it was investigated whether partial 16S rDNA sequences are sufficient to determine species identity. Sequence alignments led to the identification of four 16S rDNA regions with high degrees of interspecies variation but with highly conserved sequence patterns within the respective species. A simple protocol based on the analysis of these sequence patterns was developed, which enabled the unambiguous identification of the majority of Campylobacter species. We recommend 16S rDNA sequence analysis as an effective, rapid procedure for the specific identification of campylobacters.
Campylobacter species are important pathogens that cause a variety of diseases in humans and animals (26, 43). The most prominent members of these proteobacteria are the species Campylobacter coli and C. jejuni, the latter of which is considered the most common cause of acute bacterial enteritis worldwide (3). To date, the genus Campylobacter comprises 16 species, and among them several species other than C. jejuni and C. coli are becoming increasingly recognized as significant human pathogens (26). However, recovery and identification of these species require specialized preparatory procedures for specimens, such as filtration steps and selective incubation methods (e.g., the use of a hydrogen-enriched atmosphere) (26). Since most routine laboratories do not use these techniques, infections caused by these taxa are likely to be underdiagnosed (13, 27). In addition, phenotypic tests have only a limited discriminatory potential for the distinctive identification of Campylobacter species. These pathogens are slowly growing, fastidious organisms and are considered biochemically unreactive. As a result, extensive identification schemes comprising up to 67 phenotypic features are used to correctly identify the entire spectrum of campylobacteria (40). Moreover, the phenotypic tests used in most routine laboratories lack standardization, although it is known that even minor parameters such as the inoculum size affect the results (39). The existence of biochemically atypical strains, which exhibit unusual phenotypic profiles, represents an additional challenge (36). In these cases not even lengthy and extensive laboratory procedures make an unequivocal identification of the respective species possible. Taken together, identification of campylobacters to the species level is a difficult and often unsuccessful task.
Ribosomal DNA sequencing has greatly facilitated the identification of bacteria, especially in the case of fastidious pathogens for which conventional methods fall short (9, 20, 24, 45). The aim of this study was to investigate the utility of 16S rRNA gene (16S rDNA) sequencing for the species-specific identification of campylobacters. For this purpose, comparisons of 135 16S rDNA sequences of all taxa of the genus Campylobacter known to exist were performed. Fifty sequences were taken directly from GenBank, and the sequences of 85 cultivated strains were determined. The advantages as well as the limitations of this strategy are discussed. In addition, a simple protocol for the rapid identification of campylobacters, based on partial sequence analysis of variable 16S rDNA regions, was established.
MATERIALS AND METHODS
Bacterial strains and growth conditions.
Eighty-five bacterial strains were cultivated to obtain their 16S rDNA sequences. Among these 85 strains, 34 were directly obtained from national culture collections (American Type Culture Collection [ATCC]; Belgian Coordinated Collection of Microorganisms, University of Ghent, Belgium [LMG]; Culture Collection, University of Göteborg, Göteborg, Sweden [CCUG]) and 51 were recently isolated field strains. The strains and their sources are given in Table 1. All strains were plated on Columbia-blood agar plates containing 5% defibrinated sheep blood (bioMerieux, Marcy l'Etoile, France) and were incubated at 37°C in a microaerophilic atmosphere (Genbox microaer; bioMerieux) for 48 h. The anaerobic species C. gracilis and C. rectus were grown under the same conditions but in an anaerophilic atmosphere (Genbox anaer; bioMerieux).
TABLE 1.
Sources of 16S rRNA sequences used in this studya
| Species | Strain | Source (if known) | GenBank accession no.b | Reference |
|---|---|---|---|---|
| C. fetus subsp. fetus (n = 15) | ATCC 27374T | Ovine, France | M65012 | |
| F-107/4132 | Bovine, Australia | AF550618 | This study | |
| F-133/4369 | Bovine, Australia | (AF550618) | This study | |
| H00/415 | Human, Austria | AF550619 | This study | |
| H97/292 | Human, Austria | (AF550619) | This study | |
| H97/343 | Human, Austria | (AF550619) | This study | |
| H99/537 | Human, Austria | (AF550619) | This study | |
| L487 | Human, Austria | (AF550619) | This study | |
| F-RK | Human, Austria | (AF550619) | This study, 25 | |
| F1-O2 | Bovine, Germany | (AF550619) | This study | |
| F2-B88 | Bovine, Austria | (AF550619) | This study | |
| F3-H88 | Bovine, Austria | (AF550619) | This study | |
| F4-S88 | Bovine, Austria | (AF550619) | This study | |
| F1-B398/2 SK | Bovine, Austria | (AF550619) | This study | |
| F-94/4256 | Ovine, Australia | (AF550619) | This study | |
| C. fetus subsp. venerealis (n = 11) | ATCC 19438T | Bovine, England | M65011 | |
| V1-O1 | Germany | (AF550619) | This study | |
| V2-O1 | Germany | (AF550619) | This study | |
| V3-O1 | Germany | (AF550619) | This study | |
| V4-D96 | Bovine, Austria | (AF550619) | This study | |
| V5-G91 | Bovine, Austria | (AF550619) | This study | |
| V6-Th15 | Bovine, Austria | (AF550619) | This study | |
| V7-Th24 | Bovine, Austria | (AF550619) | This study | |
| V-80/4172 | Bovine, Australia | (AF550619) | This study | |
| V-108/4111 | Bovine, Australia | (AF550619) | This study | |
| V-121/4401 | Bovine, Australia | (AF550619) | This study | |
| C. coli (n = 11) | LMG 9220 | Human, Belgium | AF550620 | This study |
| LMG 15883 | Porcine, Australia | AF550621 | This study | |
| LMG 15884 | Porcine, Australia | AF550622 | This study | |
| H99/119 | Human, Austria | AF550623 | This study | |
| H99/164 | Human, Austria | (AF550623) | This study | |
| B99/222 | Human, Austria | (AF550623) | This study | |
| H99/155 | Human, Austria | AF550624 | This study | |
| B99/131 | Human, Austria | AF550625 | This study | |
| B99/90 | Human, Austria | (AF550625) | This study | |
| ATCC 33559T, CCUG 11283c | Porcine, Belgium | M59073, L04312 | ||
| RMIT32A | L19738 | |||
| C. jejuni subsp. jejuni (n = 14) | LMG 9217 | Human, Belgium | AF550626 | This study |
| B99/207 | Human, Austria | (AF550626) | This study | |
| B99/210 | Human, Austria | (AF550626) | This study | |
| B99/223 | Human, Austria | (AF550626) | This study | |
| H99/241 | Human, Austria | (AF550626) | This study | |
| H99/240 | Human, Austria | AF550628 | This study | |
| B99/224 | Human, Austria | AF550629 | This study | |
| B99/206 | Human, Austria | AF550630 | This study | |
| H99/244 | Human, Austria | (AF550630) | This study | |
| H99/246 | Human, Austria | (AF550630) | This study | |
| H99/245 | Human, Austria | (AF550630) | This study | |
| ATCC 33560T, CCUG 11284c | Bovine | M59298, L04315 | ||
| NCTC 11168 | Human | AL111168 | ||
| ATCC 43431 | Human | Z29326 | ||
| C. jejuni subsp. doylei (n = 3) | LMG 9243 | Human, Belgium | AF550627 | This study |
| CCUG 24567 | Human, Australia | L14630 | ||
| SS1-5384-98 | Y19244 | |||
| C. lari (n = 6) | LMG 7607 | Human | AF550631 | This study |
| LMG 11251 | Belgium | AF550632 | This study | |
| LMG 11760 | Human, Canada | AF550633 | This study | |
| LMG 14338 | Human, Belgium | AF550634 | This study | |
| CCUG23947T | Avian | L04316 | ||
| CF89-12 | AB066098 | |||
| C. sputorum (n = 9) | LMG 10388 | Ovine, Sweden | AF550635 | This study |
| LMG 11761 | Human, Canada | AF550636 | This study | |
| LMG 6617 | Ovine | AF550637 | This study | |
| CSP-1 | Bovine, Austria | AF550638 | This study | |
| CSP-2 | Bovine, Austria | AF550639 | This study | |
| CSP-3 | Bovine, Austria | (AF550639) | This study | |
| ATCC 33491 | L04319 | |||
| BU-112B | AF022768 | |||
| LMG7795T | X67775 | /PICK> | ||
| C. upsaliensis (n = 10) | LMG 8851 | Human, United Kingdom | AF550640 | This study |
| LMG 7915 | Human, United States | (AF550640) | This study | |
| SK H5 | Canine, Germany | (AF550640) | This study, 33 | |
| J31.000 | Human, Austria | (AF550640) | This study | |
| LMG 8853 | Human, Australia | AF550641 | This study | |
| H29 | Canine, Germany | AF550642 | This study, 33 | |
| H37 E/1 | Canine, Germany | AF550643 | This study, 33 | |
| H53 E/ccda | Canine, Germany | AF550644 | This study, 33 | |
| L461 | Human, Austria | AF550645 | This study | |
| CCUG 14913 | Canine, Sweden | L14628 | ||
| C. helveticus (n = 4) | LMG 12639 | Feline, Switzerland | AF550646 | This study |
| CCUG 30563 | Feline, Switzerland | AF550647 | This study | |
| CCUG34016 | Feline, Sweden | AF550648, AF550649 | This study | |
| NCTC 12470T | Feline | U03022 | ||
| C. curvus (n = 5) | LMG 11034 | Human, United States | AF550650 | This study |
| LMG 11127 | Belgium | AF550651 | This study | |
| LMG 13936 | Human, Belgium | AF550652 | This study | |
| ATCC 35224T | Human, United States | L04313 | ||
| C10ETOH | L06976 | |||
| C. concisus (n = 5) | LMG 7545 | Human, Sweden | AF550653 | This study |
| LMG 7961 | Human, Sweden | (AF550653) | This study | |
| LMG 13937 | Human, Belgium | AF550654 | This study | |
| ATCC 33237T | Human | L04322 | ||
| FDC 288 Tanner | L06977 | |||
| C. showae (n = 3) | LMG 12636 | Human, United States | AF550655 | This study |
| CCUG 11641 | Human, Sweden | L06975 | ||
| CCUG 30254T | Human, United States | L06974 | ||
| C. gracilis (n = 5) | LMG 7616 | United States | AF550656 | This study |
| CCUG 13143 | Human, United States | AF550657 | This study | |
| CCUG 27721 | United States | AF550658 | This study | |
| ATCC 33236T | Human, United States | L04320 | ||
| L37787 | ||||
| C. rectus (n = 4) | LMG 7611 | Human, Sweden | AF550659 | This study |
| LMG 7612 | Human, Sweden | (AF550659) | This study | |
| ATCC 33238T | Human | L04317 | ||
| CCUG 19168 | Human, Sweden | L06973 | ||
| C. mucosalis (n = 5) | ATCC 49352 | Porcine, Scotland | AF550660 | This study |
| ATCC 49353 | Porcine, Scotland | AF550661 | This study | |
| ATCC 49354 | Porcine, Scotland | AF550662 | This study | |
| ATCC 43265 | Porcine | AF550663 | This study | |
| CCUG 6822T | Porcine | L06978 | ||
| C. hyointestinalis subsp. hyointestinalis (n = 10) | H01/12 | Human, Austria | (AF097681) | This study |
| ATCC35217 | Porcine, United States | M65010 | ||
| NCTC11608T | Porcine, United States | AF097689 | 19 | |
| SVS 3038 | Porcine, Denmark | AF097691 | 19 | |
| SCRL 0425 | Bovine, Scotland | AF097681 | 19 | |
| SCRL 0943 | Bovine, Scotland | AF097682 | 19 | |
| SVS 3035 | Porcine, Denmark | AF097690 | 19 | |
| MGH 97-2652 | AF219235 | |||
| H00/108 | Human, Austria | AF499005 | 17 | |
| NADC 2006 | M65009 | |||
| C. hyointestinalis subsp. lawsonii (n = 6) | CCUG 27631 | Porcine, Sweden | AF097683 | 19 |
| CHY 4 | Porcine, England | AF097684 | 19 | |
| CHY 5 | Porcine, England | AF097685 | 19 | |
| CHY 6 | Porcine, England | AF097686 | 19 | |
| CHY 7 | Porcine, England | AF097687 | 19 | |
| CHY 8 | Porcine, England | AF097688 | 19 | |
| C. lanienae (n = 5) | S-K FAVW | Porcine, Austria | AF550664 | This study |
| NCTC 13004T | Human, Switzerland | AF043425 | 30 | |
| UB 994 | Human, Switzerland | AF043424 | 30 | |
| UB 993 | Human, Switzerland | AF043423 | 30 | |
| UB 992 | Human, Switzerland | AF043422 | 30 | |
| C. hominis (n = 4) | NCTC 13146T | Human, England | AJ251584 | |
| HS-C | Human, England | AF062492 | ||
| HS-B | Human, England | AF062491 | ||
| HS-A | Human, England | AF062490 |
A total of 135 sequences were tested.
The 16S rDNA sequences submitted to GenBank are given in boldface. Campylobacter strains sharing identical sequences are indicated by parentheses and were not submitted to GenBank.
Synonymous strains derived from different culture collections.
Species characterization.
The identities of bacterial strains obtained from culture collections were verified by Gram staining and microscopy. Species-specific identification of field strains was performed by standard phenotypic tests, as described in the literature (26, 34, 36). Strains were initially analyzed for the following properties: Gram negativity, spiral morphology, and microaerophilic growth dependency. Assays for oxidase and catalase activity as well as hippurate and indoxyl acetate hydrolysis were performed. Antimicrobial susceptibility tests with ciprofloxacin, nalidixic acid, erythromycin, tetracycline, and cephalothin were done as described recently (17). Each isolate was additionally analyzed with the API Campy system (bioMerieux). Subsequently, a more detailed characterization was performed, as needed. Subspecies of C. fetus were differentiated by testing their tolerance to 1% glycine, their ability to reduce selenite (2), and a subspecies-specific PCR method (22). C. hyointestinalis was further analyzed by whole-cell protein analysis (41), as described recently (17). Characterization of C. upsaliensis was performed as reported elsewhere (33). C. jejuni and C. coli strains were additionally characterized by species-specific PCR assays (8, 16, 29). Subspecies of C. jejuni were differentiated by their ability to reduce nitrate (26, 34). C. lanienae was characterized by the phenotypic tests described by Logan et al. (30).
DNA extraction and 16S rDNA sequencing.
PCR amplification of the 16S rRNA genes and direct sequencing of the PCR products were performed as described previously (17). To avoid the potential problem of sequence data variation due to nucleotide misincorporation by the amplifying polymerase, a high concentration of template DNA was used in the reaction mixture, as recommended elsewhere (6). Briefly, the DNAs of the bacterial strains were rapidly isolated by using Chelex 100 resin (Bio-Rad, Hercules, Calif.) (51). Three overlapping 16S rRNA gene fragments were generated by PCR in separate reactions by using the oligonucleotide primer pairs Ps5/1 (5′-TATGGAGAGTTTGATCCTGG-3′) and Ps3/1 (5′-GTTAAGCTGTTAGATTTCAC-3′), Ps5/2 (5′-AGCGTTACTCGGAATCACTG-3′) and Ps3/2 (5′-ACAGCCGTGCAGCACCTGTC-3′), and Ps5/3 (5′-AACCTTACCTGGGCTTGATA-3′) and Ps3/3 (5′-AAGGAGGTGATCCAGCCGCA-3′). Both strands of the purified PCR products were submitted to the cycle sequencing reaction with the BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, Calif.). Products were resolved on an ABI Prism 310 automated sequencer (Applied Biosystems). To facilitate detection of sequence variation, additional oligonucleotide primers were applied to amplify the variable 16S rDNA regions (Vc regions). Primers Vc5/6-F (5′-AAAGCGTGGGGAGCAAACAG-3′) and Vc5/6-R (5′-ACTTAACCCAACATCTCACG-3′) were used to amplify a 334-bp DNA fragment containing the variable regions Vc5 and Vc6. Primers Vc1/2-F (5′-AGAGTTTGATCCTGGCTCAG-3′) and Vc1/2-R (5′-TGATCATCCTCTCAGACCAG-3′) were used to amplify a 300-bp DNA fragment containing the variable regions Vc1 and Vc2. The positions of the PCR primer sequences along the Campylobacter 16S rDNA are illustrated schematically in Fig. 1.
FIG. 1.
Schematic representation of the positions of the PCR primers and the lengths of the amplicons along the Campylobacter 16S rRNA gene (≈1,500 bp). The locations of the variable regions (Vc) are indicated as shaded boxes. The location of an IVS present in several Campylobacter strains (14, 28, 48) is indicated at the top.
Cloning procedure.
The distinct PCR products generated from strain C. helveticus CCUG 34016 with primers Ps5/1 and Ps3/1 were ligated into pSTBlue-1 vector DNA by using the AccepTor Vector kit, according to the specifications of the manufacturer (Novagene, Madison, Wis.). Escherichia coli XL-1 cells were transformed (E. coli Pulser; Bio-Rad, Hercules, Calif.) with the ligation products and spread onto Luria-Bertani agar plates (42) containing 100 μg of ampicillin per ml, 20 μg of 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside per ml, and 0.1 mM isopropyl-β-d-thiogalactopyranoside. Randomly picked white colonies were analyzed for the presence of the correct inserts by colony PCR with primers T7 and SP6, as recommended by the manufacturer (Novagene). The correct identities of the fragments were confirmed by DNA sequencing.
Data analysis.
16S rDNA sequence analysis was performed with the SEQLAB program from the Wisconsin Package (version 10.2; Genetics Computer Group; Madison, Wis.) and Clustal X (version 1.81) (47). All sequences were edited to a common length representing nearly the full length of the gene (94%; nucleotides 39 to 1455, according to the C. jejuni ATCC 43431 sequence [23]). Intervening sequences present in some strains of the species C. rectus, C. sputorum, C. curvus, and C. helveticus were annotated according to criteria described elsewhere (14, 28, 48) and excised from the sequence data. The edited sequences were aligned by using the Clustal X program. Subsequently, a distance matrix was calculated from the aligned sequences by using the DISTANCES program from SEQLAB without correction for multiple base pair substitutions (uncorrected distance) (see Table 2). Neighbor-joining trees were constructed from these data with the GROWTREE program of SEQLAB and the NJPLOT program distributed with Clustal X (Fig. 2).
TABLE 2.
Homology matrix
| Species | % Variationa
|
|||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | |
| 1. C. hominis (n = 4) | 0-0.6 | 6.4 ± 0.31 | 7.6 ± 0.16 | 6.9 ± 0.16 | 7.3 ± 0.16 | 7.7 ± 0.24 | 7.1 ± 0.03 | 8.8 ± 0.04 | 8.3 ± 0.04 | 9.2 ± 0.21 | 9.5 ± 0.08 | 11.3 ± 0.03 | 11.1 ± 0.08 | 10.0 ± 0.06 | 9.7 ± 0.10 | 9.6 ± 0.06 |
| 2. C. gracilis (n = 5) | 6.0-6.8 | 0-0.1 | 7.3 ± 0.04 | 4.9 ± 0.14 | 5.7 ± 0.08 | 6.1 ± 0.23 | 5.7 ± 0.08 | 6.6 ± 0.08 | 6.3 ± 0.07 | 7.9 ± 0.56 | 7.9 ± 0.09 | 10.0 ± 0.06 | 9.9 ± 0.12 | 9.4 ± 0.08 | 9.3 ± 0.18 | 9.4 ± 0.07 |
| 3. C. sputorum (n = 8) | 7.4-7.8 | 7.2-7.4 | 0-0.1 | 6.4 ± 0.03 | 6.5 ± 0.13 | 5.6 ± 0.22 | 6.6 ± 0.03 | 7.8 ± 0.05 | 8.0 ± 0.05 | 8.5 ± 0.27 | 8.8 ± 0.14 | 9.1 ± 0.05 | 8.9 ± 0.08 | 9.2 ± 0.08 | 8.7 ± 0.22 | 8.8 ± 0.07 |
| 4. C. rectus (n = 4) | 6.7-7.1 | 4.7-5.2 | 6.4-6.4 | 0-0.9 | 2.1 ± 0.15 | 3.9 ± 0.24 | 4.6 ± 0.24 | 6.0 ± 0.27 | 6.0 ± 0.15 | 7.0 ± 0.30 | 7.8 ± 0.26 | 9.0 ± 0.06 | 8.9 ± 0.14 | 7.9 ± 0.24 | 7.7 ± 0.29 | 7.8 ± 0.11 |
| 5. C. showae (n = 3) | 7.1-7.5 | 5.6-5.9 | 6.3-6.7 | 1.8-2.3 | 0.1-0.3 | 3.9 ± 0.28 | 5.7 ± 0.07 | 6.9 ± 0.14 | 7.1 ± 0.12 | 8.3 ± 0.24 | 8.8 ± 0.16 | 9.9 ± 0.06 | 9.8 ± 0.14 | 9.1 ± 0.20 | 8.8 ± 0.29 | 8.9 ± 0.14 |
| 6. C. curvus (n = 5) | 7.5-8.1 | 5.9-6.6 | 5.4-6.0 | 3.5-4.3 | 3.5-4.4 | 0-1.1 | 3.5 ± 0.28 | 5.4 ± 0.32 | 6.1 ± 0.23 | 6.7 ± 0.34 | 6.7 ± 0.25 | 8.7± 0.28 | 8.6 ± 0.34 | 8.3 ± 0.28 | 7.8 ± 0.36 | 7.9 ± 0.27 |
| 7. C. concisus (n = 5) | 7.0-7.1 | 5.6-5.9 | 6.5-6.6 | 4.2-4.8 | 5.6-5.8 | 3.2-4.0 | 0-0.4 | 4.2 ± 0.11 | 4.9 ± 0.11 | 5.9 ± 0.49 | 5.9 ± 0.10 | 8.4 ± 0.08 | 8.3 ± 0.12 | 6.9 ± 0.11 | 6.8 ± 0.21 | 7.0 ± 0.08 |
| 8. C. mucosalis (n = 5) | 8.7-8.8 | 6.5-6.8 | 7.7-7.9 | 5.4-6.2 | 6.6-7.1 | 5.0-6.0 | 4.0-4.4 | 0.1-0.3 | 3.4 ± 0.06 | 5.2 ± 0.69 | 4.5 ± 0.15 | 8.0 ± 0.07 | 7.4 ± 0.13 | 6.5 ± 0.09 | 6.4 ± 0.18 | 6.5 ± 0.08 |
| 9. C. fetus (n = 26) | 8.2-8.4 | 6.3-6.6 | 8.0-8.2 | 5.8-6.3 | 7.0-7.4 | 5.9-6.7 | 4.7-5.2 | 3.3-3.6 | 0-0.2 | 3.1 ± 1.07 | 3.5 ± 0.11 | 7.6 ± 0.06 | 7.4 ± 0.08 | 6.1 ± 0.10 | 5.8 ± 0.32 | 6.0 ± 0.10 |
| 10. C. hyointestinalis (n = 16) | 8.8-9.5 | 7.3-9.0 | 7.9-9.0 | 6.2-7.6 | 7.6-8.8 | 5.9-7.6 | 5.0-6.7 | 4.3-6.4 | 1.6-4.7 | 0-4.5 | 2.7 ± 0.43 | 6.7 ± 0.47 | 7.4 ± 0.25 | 5.6 ± 0.38 | 5.1 ± 0.57 | 5.3 ± 0.49 |
| 11. C. lanienae (n = 5) | 9.4-9.6 | 7.7-8.1 | 8.6-9.0 | 7.3-8.1 | 8.6-9.1 | 6.3-7.1 | 5.7-6.0 | 4.2-4.7 | 3.4-3.8 | 1.9-3.4 | 0-0.9 | 6.3 ± 0.11 | 7.3 ± 0.19 | 5.1 ± 0.18 | 4.6 ± 0.35 | 4.9 ± 0.11 |
| 12. C. helveticus (n = 4) | 11.2-11.3 | 9.9-10.1 | 9.0-9.1 | 8.9-9.1 | 9.8-10.0 | 8.5-9.3 | 8.2-8.5 | 7.9-8.1 | 7.5-7.8 | 5.9-7.5 | 6.2-6.5 | 0.1-0.3 | 2.0 ± 0.39 | 3.9 ± 0.23 | 3.6 ± 0.27 | 3.4 ± 0.11 |
| 13. C. upsaliensis (n = 10) | 10.9-11.2 | 9.8-10.2 | 8.8-9.1 | 8.6-9.2 | 9.5-10.2 | 8.2-9.7 | 8.0-8.6 | 7.1-7.7 | 7.2-7.7 | 7.0-8.1 | 7.0-7.9 | 1.6-3.2 | 0-1.3 | 4.9 ± 0.26 | 4.5 ± 0.32 | 4.3 ± 0.16 |
| 14. C. lari (n = 6) | 9.9-10.0 | 9.3-9.5 | 9.0-9.3 | 7.5-8.3 | 8.9-9.6 | 8.0-9.0 | 6.7-7.1 | 6.3-6.6 | 5.9-6.4 | 4.6-6.2 | 4.6-5.4 | 3.3-4.1 | 4.2-5.2 | 0-2.5 | 1.6 ± 0.68 | 1.5 ± 0.58 |
| 15. C. coli (n = 11) | 9.5-9.8 | 8.9-9.6 | 8.1-9.0 | 6.9-8.1 | 8.1-9.3 | 7.0-8.6 | 6.2-7.1 | 5.9-6.7 | 5.0-6.3 | 3.5-6.1 | 3.5-5.0 | 3.3-4.4 | 4.1-5.4 | 0.5-3.1 | 0-1.5 | 0.4 ± 0.42 |
| 16. C. jejuni (n = 17) | 9.5-9.8 | 9.3-9.7 | 8.6-9.0 | 7.6-8.1 | 8.7-9.3 | 7.6-8.5 | 6.9-7.2 | 6.4-6.8 | 5.9-6.4 | 4.5-6.2 | 4.7-5.2 | 3.1-3.6 | 4.0-4.8 | 0.6-2.3 | 0.0-1.6 | 0-0.4 |
The numbers above the diagonal are mean values of percent 16S rDNA variation and the respective standard deviation. The numbers below the diagonal (in boldface) are the range of 16S rDNA variation (in percent) among Campylobacter species. The numbers in the subheads correspond to the numbers for the species listed on the left.
FIG. 2.
Dendrogram of Campylobacter strains calculated from data for the nearly complete (94%) 16S rDNA sequence. Analysis placed most sequences into species-specific clusters (shaded boxes). Strains which deviated from the species-specific clustering are indicated by asterisks. The scale bar at the top indicates a 1% difference in nucleotide sequence.
Statistical calculations.
SigmaStat software (version 2.03; SPSS Inc., Chicago, Ill.) was used for statistical analysis. Either the t test or the Mann-Whitney rank sum test was used to test for the significance of differences of 16S rDNA variations among Campylobacter species.
Nucleotide sequence accession numbers.
The accession numbers of the 50 16S rDNA sequences obtained from GenBank are listed in Table 1. The unique 16S rDNA sequences (n = 47) which were derived from cultivated strains have been deposited in GenBank. Their respective accession numbers are also listed in Table 1.
RESULTS
Sequence analysis of 16S rDNAs.
The objective of this study was to determine whether 16S rDNA sequencing is a reliable approach for the specific identification of Campylobacter species. Three sets of oligonucleotide primers were used to generate sequences encompassing nearly the full length of the 16S rRNA gene. A minimum of 94% of the complete 16S rDNA (ranging from 1,415 to 1,419 bp) was obtained from all strains analyzed. Intervening sequences (IVSs) were detected within the 16S rRNA genes of 12 strains. Six strains of C. sputorum harbored IVSs of either 232 bp (CSP-1, LMG 10388, LMG 11761, LMG 6617) or 231 bp (CSP-2, CSP-3). Two strains of C. rectus (LMG 7611, LMG 7612) had IVSs of 189 bp, and three strains of C. curvus (LMG 11034, LMG 11127, LMG 13936) contained IVSs of 140 bp. Two different 16S rDNA sequences appeared to be present in strain C. helveticus CCUG 34016, as primer pair Ps5/1 and Ps3/1 generated two different amplification products of 595 and 740 bp (data not shown). Cloning and sequence analysis of these products revealed that the larger 16S rDNA fragment contained an IVS of 145 bp, whereas the smaller fragment lacked this IVS. All observed IVSs were inserted following base position 213 of the C. jejuni (ATCC 43431) 16S rDNA (23).
16S rDNA sequence diversity.
In order to evaluate whether 16S rDNA data reliably determine species identity and sufficiently discriminate among Campylobacter species, it was necessary to calculate the amount of intraspecies and interspecies 16S rDNA sequence variation. Multiple alignments of 135 Campylobacter 16S rDNA sequences of all taxa of the genus known to exist were performed. A matrix representing the sequence variations among the strains analyzed was calculated. Subsequently, a dendrogram was constructed from these data to verify the species-specific cluster formation of the sequences.
The maximum intraspecies 16S rDNA sequence diversities ranged from 0.1 to 0.9% when multiple strains of the same species were compared (Table 2). Higher degrees of maximum intraspecies diversity were found within the taxa C. curvus (1.1%), C. upsaliensis (1.3%), C. lari (2.5%), C. coli (1.5%), and C. hyointestinalis (4.5%). For the last species, a higher level of variation was attributed to a significant difference in 16S rDNA sequences between the two subspecies of C. hyointestinalis (C. hyointestinalis subsp. hyointestinalis and C. hyointestinalis subsp. lawsonii). These subspecies displayed a mean ± standard deviation sequence diversity of 3.7% ± 0.84%. This allowed the subspecies-specific differentiation of 14 of 15 strains, as displayed by the dendrogram in Fig. 2. Strain C. hyointestinalis subsp. hyointestinalis SVS 3038 (GenBank accession no. AF097691) was the sole exception. The 16S rDNA sequence of that strain exhibited a minimum of only 1% diversity from the sequence of C. hyointestinalis subsp. lawsonii and, therefore, clustered together with that subspecies (Fig. 2). The 16S rDNA variations were not adequate to differentiate between the subspecies of C. jejuni or between the subspecies of C. fetus, since in both cases several strains of each subspecies had sequences identical to those of strains of the other subspecies.
The minimum interspecies 16S rDNA sequence diversities ranged from 0 to 11.2% (Table 2). To gain a detailed view of whether 16S rDNA data reliably discriminate among the taxa, the sequence diversities of all 135 strains were visualized by a dendrogram analysis. Cluster analysis placed most sequences into groups that correlated with species (Fig. 2). The exception from these findings was a lack of discrimination among the taxa C. coli and C. jejuni and two C. lari strains. Several C. coli and C. jejuni strains had identical 16S rDNA sequences (e.g., C. coli H99/119 and C. jejuni LMG 9217), and nearly all C. coli and C. jejuni strains were assigned to a common cluster. Only two strains of C. coli, type strain CCUG 11238 and strain LMG 9220, displayed higher degrees of variation and could therefore be clearly distinguished from C. jejuni (Fig. 2). In addition, two strains of C. lari, CF89-12 and LMG 11760, were also assigned to the cluster that comprised C. coli and C. jejuni due to highly related 16S rDNA sequences (Fig. 2). Both strains displayed atypical phenotypic profiles not consistent with classical nalidixic-acid resistant thermophilic C. lari (NARTC). Strain CF89-12 was a urease-producing thermophilic C. lari strain, and strain LMG 11760 was a nalidixic acid-susceptible C. lari strain. Their minimal sequence diversities from the sequences of C. coli (0.5%) and C. jejuni (0.6%) were significantly different from those of the classical C. lari strains compared to the sequences of C. coli and C. jejuni (1.6%) (P < 0.001). The 16S rDNA sequence diversities among Campylobacter species are given in Table 2.
Characterization of variable regions within the 16S rDNA.
To improve the analysis, we investigated whether particular regions of 16S rDNA yield sufficient information to discriminate among the taxa. 16S rDNA alignment studies revealed four variable gene regions, which were termed Vc6, Vc5, Vc2, and Vc1, in accordance with the variable regions of the procaryotic 16S rRNA. These regions displayed a high level of interspecies sequence variation. Among these we discerned several sequence patterns that are applicable for species-specific identification. Figures 3A to D show the alignments of the Campylobacter 16S rDNA sequences corresponding to the Vc regions. Campylobacter species were grouped according to the particular sequence patterns within the respective Vc regions. Five distinct patterns, termed 6A to 6E, were found in the Vc6 region (Fig. 3A). Seven patterns, termed 5A to 5G, were defined in Vc5 (Fig. 3B). Twelve patterns, termed 2A to 2L, were defined in Vc2 (Fig. 3C). Analysis of Vc1 revealed eight patterns, termed 1A to 1H (Fig. 3D). These patterns were themselves species specific, or alternatively, specific variations within a general DNA motif could be ascribed to one or more species. Discrimination in the latter case required comparison of partial sequence data from more than one Vc region (see below).
FIG. 3.
(A) Alignment of campylobacter 16S rDNA sequences within the Vc6 region revealed five distinct sequence patterns (patterns 6A to 6E). Only nucleotides different from those of C. fetus (pattern 6A) are indicated. (B) Alignment of campylobacter 16S rDNA sequences within the Vc5 region revealed seven distinct sequence patterns (patterns 5A to 5G). Only nucleotides different from those of C. fetus (pattern 5A) are indicated. (C) Alignment of campylobacter 16S rDNA sequences within the Vc2 region revealed 12 distinct sequence patterns (patterns 2A to 2L). Only nucleotides different from those of C. fetus (pattern 2A) are indicated. (D) Alignment of campylobacter 16S rDNA sequences within the Vc1 region revealed eight distinct sequence patterns (patterns 1A to 1H). Only nucleotides different from those of C. fetus (pattern 1A) are indicated. Dashes indicate deletions at the respective base position. (A to D) a, nucleotide positions corresponding to the E. coli 16S rRNA (5); b, nucleotides and positions of infrequently occurring polymorphisms within the sequence pattern.
Identification scheme for campylobacters based on partial 16S rDNA analysis.
The distinct sequence patterns of the Vc regions were used to develop a simplified scheme for the species-specific identification of campylobacters by partial 16S rDNA analysis. As shown in Table 3, most species displayed a unique panel of DNA patterns, which enabled their unambiguous identification. The exception was a lack of discrimination among strains of C. jejuni and C. coli and atypical C. lari strains (CF89-12, LMG 11760), which shared the pattern 6D-5D-2E-1D. In addition, strains of C. hyointestinalis and C. lanienae, which displayed the pattern 6D-5B/5C-2C-1B, could not be discriminated.
TABLE 3.
Pattern distribution among Campylobacter species
| Species | Variable region
|
|||
|---|---|---|---|---|
| Vc6 | Vc5 | Vc2 | Vc1 | |
| C. fetus | 6A | 5A | 2A | 1A |
| C. hyointestinalis | 6B/6D | 5A/5B/5C | 2A/2B/2C | 1A/1B |
| C. lanienae | 6D | 5B/5C | 2C | 1B |
| C. mucosalis | 6C | 5C | 2C | 1C |
| C. upsaliensis | 6C | 5D | 2D | 1D |
| C. coli | 6D | 5D/5B | 2E | 1D |
| C. jejuni | 6D | 5D | 2E | 1D |
| C. lari | 6D | 5D | 2E | 1F/1Da |
| C. helveticus | 6D | 5D | 2D | 1D |
| C. curvus | 6E | 5C | 2F | 1B/1Cb |
| C. sputorum | 6E | 5E | 2G | 1G |
| C. concisus | 6E | 5C | 2H | 1B |
| C. rectus | 6E | 5F | 2I | 1C |
| C. showae | 6E | 5F | 2J | 1C |
| C. gracilis | 6E | 5E | 2K | 1E |
| C. hominis | 6E | 5G | 2L | 1H |
Strains LMG 11760 and CF89-12.
Strain C10ETHO.
DISCUSSION
The unambiguous identification of Campylobacter species is difficult because these pathogens are slowly growing, fastidious organisms which display only a few differential phenotypic properties (36). Since automated DNA sequencing has become generally available and the contents of public sequence databases are constantly increasing, 16S rDNA analysis has become a valuable tool for determination of the identities of bacterial isolates (9, 18, 20, 24, 31). Therefore, we focused on 16S rDNA sequencing to investigate its utility for the species-specific identification of campylobacters.
Present guidelines suggest that 3% variation between two rDNAs is the threshold at which two strains may be considered to represent distinct species (7, 15, 24, 44). By taking this value of sequence variation into account, the data derived from our analysis of the whole-gene sequences is summarized as follows. (i) Most Campylobacter species could clearly be differentiated, since the minimum 16S rDNA sequence variation among the most related taxa exceeded the 3% threshold (Table 2). (ii) Lower levels of 16S rDNA variations were found between the species C. rectus and C. showae (minimum diversity, 1.8%), C. hyointestinalis and C. lanienae (minimum diversity, 1.9%), C. helveticus and C. upsaliensis (minimum diversity, 1.6%), C. hyointestinalis subsp. hyointestinalis and C. fetus (minimum diversity, 1.6%), and classical (NARTC) C. lari strains and C. jejuni-C. coli (minimum diversity, 1.6%). Nevertheless, in all of these cases the interspecies variation significantly exceeded the intraspecies variation (P < 0.001) and the dendrogram analysis revealed a species-specific clustering (Fig. 2). We conclude that 16S rDNA-based differentiation of these species displaying sequence diversities below 3% has practical application. (iii) The limitation of the 16S rDNA analysis is the inability to differentiate the species C. jejuni and C. coli and atypical C. lari strains. Several C. jejuni and C. coli strains shared identical 16S rDNA sequences, and nearly all strains of these taxa were assigned to a common cluster (Fig. 2). In addition, two atypical C. lari strains analyzed in this study were also assigned to this cluster (Fig. 2). Their 16S rDNA sequences displayed minimum diversities of 0.5% compared to the sequences of C. coli and 0.6% compared to the sequences of C. jejuni, whereas the maximum intraspecies diversity of C. coli was 1.5% and that of C. jejuni was 0.4%. In contrast, classical (NARTC) C. lari strains displayed higher degrees of variation and could therefore be differentiated from this cluster (Fig. 2). The observations that the members of the species C. lari are phenotypically and genotypically diverse and that the species may comprise multiple taxa are in concordance with the findings presented in several other reports and highlight the fact that the taxonomy of C. lari is still in progress (4, 10, 11, 12, 32, 37). Since C. jejuni, C. coli, and C. lari are significant pathogens and their differentiation is important when they are involved in clinical cases of infection, we suggest the use of recently described PCR assays for accurate discrimination and identification of the respective taxon (16, 29, 49, 50).
The identification of taxa to the subspecies level was possible for 14 of 15 strains of C. hyointestinalis. The exception was strain C. hyointestinalis subsp. hyointestinalis SVS 3038, which demonstrated a clear phylogenetic affinity with C. hyointestinalis subsp. lawsonii strains, as described recently (19). Since the taxonomic status of this strain remains unclear, we cannot recommend 16S rDNA analysis as a singular method for the differentiation of C. hyointestinalis subspecies. A polyphasic approach that uses both phenotypic and genotypic methods should be used for identification of the subspecies (19). Subspecies-specific identification of the taxa C. jejuni and C. fetus by 16S rDNA analysis was not possible (38).
This study further shows that improved differentiation is possible by modification of 16S rDNA analysis. For this purpose partial sequence data were used to determine species identities. The general structures of 16S rRNAs and rDNAs comprise highly conserved and variable regions. Sequence alignments of the Campylobacter 16S rDNA operon revealed four highly variable regions, termed Vc6, Vc5, Vc2, and Vc1. These regions represent the highly variable areas V6 (Vc6), V5 (Vc5), V2 (Vc2), and V1 (Vc1) of the procaryotic 16S rRNA (rDNA) (35). The sequences of the Vc regions exhibited high levels of diversity among the different Campylobacter species but displayed fixed patterns within the species themselves. Nearly all Campylobacter species displayed characteristic sequence patterns and could be clearly discriminated (Table 3). The exception was a lack of differentiation among the taxa C. coli and C. jejuni and atypical C. lari isolates, which had already been revealed by complete 16S rDNA analysis. Analysis of the Vc regions indicated the pattern 6D-5D-2E-1D for these taxa and the two C. lari isolates. To ensure clear differentiation, we recommend PCR assays of the other gene sequences mentioned above. In addition, discrimination of certain isolates of C. hyointestinalis and C. lanienae, which displayed the pattern 6D-5B/5C-2C-1B, was also not possible. In these cases, however, discrimination is achieved by complete 16S rDNA sequence analysis.
Moreover, it is significant that complete 16S rDNA as well as analysis of the Vc regions can be used to discriminate closely related taxa, such as Bacteroides ureolyticus or Helicobacter and Arcobacter, from Campylobacter species. This is important, since these pathogens possess few phenotypic criteria which could serve as useful markers for their unambiguous identification. For instance, both Helicobacter pullorum and Arcobacter butzleri have habitats (e.g., pigs and chicken) and disease associations (e.g., gastroenteritis) similar to those of several campylobacters, contributing to their misidentification as campylobacters by conventional phenotypic tests (1, 21, 36, 46, 52).
We conclude that comparisons of 16S rDNA sequences provide a substantially improved basis for the identification and differentiation of campylobacter species. Focused analysis of the variable regions offers the ability to identify nearly the same range of species as whole-gene analysis, however, with the advantages of higher efficiency and lower cost. Although the significant pathogens C. jejuni, C. coli, and C. lari cannot be reliably discriminated by use of the 16S rDNA data, the approach reported here offers obvious advantages over existing methods. At present, no other singular method has the ability to identify such an extensive range of Campylobacter species. Furthermore, identification and differentiation are achieved within 2 days, in contrast to standard biochemical identification, which may take more than a week or which may even fail to provide reliable results for certain strains. The addition of 47 campylobacter sequences to the database should prove valuable for clinical microbiologists using 16S rDNA-based analysis during routine identification. In addition, we expect that the detailed description of the variable 16S rDNA regions provided here will facilitate the design of species-specific probes, PCR assays, and oligonucleotide arrays, which will further improve the ability to identify campylobacters from various specimens.
Acknowledgments
We are grateful to Karl Bauer, Klemens Fuchs, and Rainer Rosegger for helpful discussions. We thank the following colleagues for providing us with Campylobacter strains: S. Hum (Camden, Australia), I. Moser (Berlin, Germany), G. Kirpal (Hannover, Germany), E. Pohl (Aulendorf, Germany), E. Hofer and J. Flatscher (Mödling, Austria), and R. Krause, B. Ursinitsch, and K. Helleman (Graz, Austria).
REFERENCES
- 1.al Rashid, S. T., I. Dakuna, H. Louie, D. Ng, P. Vandamme, W. Johnson, and V. L. Chan. 2000. Identification of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, Arcobacter butzleri, and A. butzleri-like species based on the glyA gene. J. Clin. Microbiol. 38:1488-1494. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Bisping, W., and G. Amtsberg. 1988. Genus: Campylobacter, p. 215-231. In Colour atlas for the diagnosis of bacterial pathogens in animals. Paul Parey Scientific Publishers, Berlin, Germany.
- 3.Blaser, M. J. 1997. Epidemiologic and clinical features of Campylobacter jejuni infections. J. Infect. Dis. 176:103-105. [DOI] [PubMed] [Google Scholar]
- 4.Bolton, F. J., D. Coates, D. N. Hutchinson, and A. F. Godfree. 1987. A study of thermophilic campylobacters in a river system. J. Appl. Bacteriol. 62:167-176. [DOI] [PubMed] [Google Scholar]
- 5.Brosius, J., M. L. Palmer, P. J. Kennedy, and H. F. Noller. 1978. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc. Natl. Acad. Sci. USA 75:4801-4805. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Cha, R. S., and W. G. Thilly. 1995. Specificity, efficiency, and fidelity of PCR, p. 37-51. In C. W. Dieffenbach and G. S. Dveksler (ed.), PCR primer: a laboratory protocol. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
- 7.Clayton, R. A., G. Sutton, P. S. Hinkle, Jr., C. Bult, and C. Fields. 1995. Intraspecific variation in small-subunit rRNA sequences in GenBank: why single sequences may not adequately represent prokaryotic taxa. Int. J. Syst. Bacteriol. 45:595-599. [DOI] [PubMed] [Google Scholar]
- 8.Denis, M., C. Soumet, K. Rivoal, G. Ermel, D. Blivet, G. Salvat, and P. Colin. 1999. Development of a m-PCR assay for simultaneous identification of Campylobacter jejuni and C. coli. Lett. Appl. Microbiol. 29:406-410. [DOI] [PubMed] [Google Scholar]
- 9.Drancourt, M., C. Bollet, A. Carlioz, R. Martelin, J.-P. Gayral, and D. Raoult. 2000. 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J. Clin. Microbiol. 38:3623-3630. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Duim, B., P. A. Vandamme, A. Rigter, S. Laevens, J. R. Dijkstra, and J. A. Wagenaar. 2001. Differentiation of Campylobacter species by AFLP fingerprinting. Microbiology 147:2729-2737. [DOI] [PubMed] [Google Scholar]
- 11.Duim, B., J. A. Wagenaar, H. P. Endtz, J. R. Dijkstra, and P. A. R. Vandamme. 2001. Identification of distinct genogroups of C. lari with AFLP and protein profile analysis. Int. J. Med. Microbiol. 291(Suppl. 31):143. [Google Scholar]
- 12.Endtz, H. P., J. S. Vliegenthart, P. Vandamme, H. W. Weverink, N. P. van den Braak, H. A. Verbrugh, and A. van Belkum. 1997. Genotypic diversity of Campylobacter lari isolated from mussels and oysters in The Netherlands. Int. J. Food Microbiol. 34:79-88. [DOI] [PubMed] [Google Scholar]
- 13.Engberg, J., S. L. W. On, C. S. Harrington, and P. Gerner-Schmidt. 2000. Prevalence of Campylobacter, Arcobacter, Helicobacter, and Suturella spp. in human fecal samples as estimated by reevaluation of isolation methods for campylobacters. J. Clin. Microbiol. 38:286-291. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Etoh, Y., A. Yamamoto, and N. Goto. 1997. Intervening sequences in the 16S rRNA genes of Campylobacter sp.: diversity of nucleotide sequences and uniformity of location. Microbiol. Immunol. 42:241-243. [DOI] [PubMed] [Google Scholar]
- 15.Fox, G. E., J. D. Wisotzkey, and P. Jurtshuk, Jr. 1992. How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. Int. J. Syst. Bacteriol. 42:166-170. [DOI] [PubMed] [Google Scholar]
- 16.Gonzalez, I., K. A. Grant, P. T. Richardson, S. F. Park, and M. D. Collins. 1997. Specific identification of the enteropathogens Campylobacter jejuni and Campylobacter coli by using a PCR test based on the ceuE gene encoding a putative virulence determinant. J. Clin. Microbiol. 35:759-763. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Gorkiewicz, G., G. Feierl, R. Zechner, and E. L. Zechner. 2002. Transmission of Campylobacter hyointestinalis from a pig to a human. J. Clin. Microbiol. 40:2601-2605. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Harmsen, D., J. Rothgänger, M. Frosch, and J. Albert. 2002. RIDOM: ribosomal differentiation of medical micro-organisms database. Nucleic Acids Res. 30:416-417. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Harrington, C. S., and S. L. W. On. 1999. Extensive 16S rRNA gene sequence diversity in Campylobacter hyointestinalis strains: taxonomic and applied implications. Int. J. Syst. Bacteriol. 49:1171-1175. [DOI] [PubMed] [Google Scholar]
- 20.Hayden, R. T., C. P. Kolbert, M. K. Hopkins, and D. H. Persing. 2001. Characterization of culture-derived spiral bacteria by 16S ribosomal RNA gene sequence analysis. Diagn. Microbiol. Infect. Dis. 39:55-59. [DOI] [PubMed] [Google Scholar]
- 21.Houf, K., A. Tutenel, L. de Zutter, J. van Hoof, and P. Vandamme. 2000. Development of a multiplex PCR assay for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii. FEMS Microbiol. Lett. 193:89-94. [DOI] [PubMed] [Google Scholar]
- 22.Hum, S., K. Quinn, J. Brunner, and S. L. W. On. 1997. Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies. Aust. Vet. J. 75:827-831. [DOI] [PubMed] [Google Scholar]
- 23.Kim, N. W., R. R. Gutell, and V. L. Chan. 1995. Complete sequences and organization of the rrnA operon from Campylobacter jejuni TGH9011 (ATCC43431). Gene 164:101-106. [DOI] [PubMed] [Google Scholar]
- 24.Kolbert, C. P., and D. H. Persing. 1999. Ribosomal DNA sequencing as a tool for identification of bacterial pathogens. Curr. Microbiol. 2:299-305. [DOI] [PubMed] [Google Scholar]
- 25.Krause, R., S. Ramschak-Schwarzer, G. Gorkiewicz, W. J. Schnedl, G. Feierl, C. Wenisch, and E. C. Reisinger. 2002. Recurrent septicemia due to Campylobacter fetus and Campylobacter lari in an immunocompetent patient. Infection 30:171-174. [DOI] [PubMed] [Google Scholar]
- 26.Lastovica, A. J., and M. B. Skirrow. 2000. Clinical significance of Campylobacter and related species other than Campylobacter jejuni and C. coli, p. 89-120. In I. Nachamkin and M. J. Blaser (ed.), Campylobacter, 2nd ed. American Society for Microbiology, Washington, D.C.
- 27.Le Roux, E., and A. J. Lastovica. 1998. The Cape Town protocol: how to isolate the most campylobacters for your dollar, pound, franc, yen, etc., p. 30-33. In A. J. Lastovica, D. G. Newell, and E. E. Lastovica (ed.), Campylobacter, Helicobacter and related organisms. Institute of Child Health, Cape Town, South Africa.
- 28.Linton, D., F. E. Dewhirst, J. P. Clewley, R. J. Owen, A. P. Burnens, and J. Stanley. 1994. Two types of 16S rRNA gene are found in Campylobacter helveticus: analysis, applications and characterization of the intervening sequence found in some strains. Microbiology 140:847-855. [DOI] [PubMed] [Google Scholar]
- 29.Linton, D., A. J. Lawson, R. J. Owen, and J. Stanley. 1997. PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli direct from diarrheic stools. J. Clin. Microbiol. 35:2568-2572. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Logan, J. M. J., A. Burnens, D. Linton, A. J. Lawson, and J. Stanley. 2000. Campylobacter lanienae sp. nov., a new species isolated from workers in an abbatoir. Int. J. Syst. E vol. Microbiol. 50:865-872. [DOI] [PubMed] [Google Scholar]
- 31.Maidak, B. L., J. R. Cole, T. G. Lilburn, C. T. Parker, Jr., P. R. Saxman, R. J. Farris, G. M. Garrity, G. J. Olsen, T. M. Schmidt, and J. M. Tiedje. 2001. The RDP-II (Ribosomal Database Project). Nucleic Acids Res. 29:173-174. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Mégraud, F., D. Chevrier, N. Desplaces, A. Sedallian, and J. L. Guesdon. 1988. Urease-positive thermophilic Campylobacter (Campylobacter laridis variant) isolated from an appendix and from human feces. J. Clin. Microbiol. 26:1050-1051. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Moser, I., B. Rieksneuwöhner, P. Lentzsch, P. Schwerk, and L. H. Wieler. 2001. Genomic heterogeneity and O-antigenic diversity of Campylobacter upsaliensis and Campylobacter helveticus strains isolated from dogs and cats in Germany. J. Clin. Microbiol. 39:2548-2557. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Nachamkin, I. 1995. Campylobacter and Arcobacter, p. 483-491. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology, 6th ed. American Society for Microbiology, Washington, D.C.
- 35.Neefs, J.-M., Y. van de Peer, L. Hendriks, and R. de Wachter. 1990. Compilation of small ribosomal subunit RNA sequences. Nucleic Acids Res. 18:2237-2317. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.On, S. L. W. 1996. Identification methods for campylobacters, helicobacters, and related organisms. Clin. Microbiol. Rev. 9:405-422. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.On, S. L. W., P. I. Fields, T. Broman, L. O. Helsel, C. Fitzgerald, C. S. Harrington, S. Laevens, A. G. Steigerwalt, B. Olsen, and P. A. R. Vandamme. 2001. Polyphasic taxonomic analysis of Campylobacter lari: delineation of three subspecies. Int. J. Med. Microbiol. 291(Suppl. 31):144. [Google Scholar]
- 38.On, S. L. W., and C. S. Harrington. 2001. Evaluation of numerical analysis of PFGE-DNA profiles for differentiating Campylobacter fetus subspecies by comparison with phenotypic, PCR and 16S rDNA sequencing methods. J. Appl. Microbiol. 90:285-293. [DOI] [PubMed] [Google Scholar]
- 39.On, S. L. W., and B. Holmes. 1991. Effect of inoculum size on the phenotypic characterization of Campylobacter species. J. Clin. Microbiol. 29:923-926. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.On, S. L. W., B. Holmes, and M. J. Sackin. 1996. A probability matrix for the identification of campylobacters, helicobacters and allied taxa. J. Appl. Microbiol. 81:425-432. [DOI] [PubMed] [Google Scholar]
- 41.Pot, B., P. Vandamme, and K. Kersters. 1994. Analysis of electrophoretic whole-organism protein fingerprints, p. 493-521. In M. Godfellow and A. G. O'Donnell (ed.), Modern microbial methods. Chemical methods in prokaryotic systematics. John Wiley & Sons, Chichester, United Kingdom.
- 42.Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
- 43.Skirrow, M. B., and M. J. Blaser. 2000. Clinical aspects of Campylobacter infection, p. 69-88. In I. Nachamkin and M. J. Blaser (ed.), Campylobacter, 2nd ed. American Society for Microbiology, Washington, D.C.
- 44.Stackebrandt, E., and B. M. Goebel. 1994. Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int. J. Syst. Bacteriol. 44:864-849. [Google Scholar]
- 45.Stackebrandt, E., W. Liesack, and D. Witt. 1992. Ribosomal RNA and rDNA sequence analyses. Gene 115:225-260. [DOI] [PubMed] [Google Scholar]
- 46.Steinbrueckner, B., G. Haerter, K. Pelz, A. Burnens, and M. Kist. 1998. Discrimination of Helicobacter pullorum and Campylobacter lari by analysis of whole cell fatty acid extracts. FEMS Microbiol Lett. 168:209-212. [DOI] [PubMed] [Google Scholar]
- 47.Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24:4876-4882. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48.van Camp, G., Y. van de Peer, S. Nicolai, J.-M. Neefs, P. Vandamme, and R. de Wachter. 1993. Structure of 16S and 23S ribosomal RNA genes in Campylobacter species: phylogenetic analysis of the genus Campylobacter and presence of internal transcribed spacers. Syst. Appl. Microbiol. 16:361-368. [Google Scholar]
- 49.van Doorn, L.-J., B. A. J. Giesendorf, R. Bax, B. A. M. van der Zeijst, P. Vandamme, and W. G. V. Quint. 1997. Molecular discrimination between Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter upsaliensis by polymerase chain reaction based on a novel putative GTPase gene. Mol. Cell. Probes 11:177-185. [DOI] [PubMed] [Google Scholar]
- 50.van Doorn, L.-J., A. Verschuuren-van Haperen, A. Burnens, M. Huysmans, P. Vandamme, B. A. J. Giesendorf, M. J. Blaser, and W. G. V. Quint. 1999. Rapid identification of thermotolerant Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis from various geographic locations by a GTPase-based PCR-reverse hybridization assay. J. Clin. Microbiol. 37:1790-1796. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 51.Walsh, P. S., D. A. Metzger, and R. Higuchi. 1991. Chelex® 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques 10:506-513. [PubMed] [Google Scholar]
- 52.Wesley, I. V., L. Schroeder-Tucker, A. L. Baetz, F. E. Dewhirst, and B. J. Paster. 1995. Arcobacter-specific and Arcobacter butzleri-specific 16S rRNA-based DNA probes. J. Clin. Microbiol. 33:1691-1698. [DOI] [PMC free article] [PubMed] [Google Scholar]