Species-Specific Identification of Campylobacters by Partial 16S ...

JOURNAL OF CLINICAL MICROBIOLOGY, June 2003, p. 2537–2546 Vol. 41, No. 6 

0095-1137/03/$08.000 DOI: 10.1128/JCM.41.6.2537–2546.2003 

Copyright © 2003, American Society for Microbiology. All Rights Reserved. 

Species-Specific Identification of Campylobacters by Partial 16S rRNA 

Gene Sequencing 

Gregor Gorkiewicz, 1 * Gebhard Feierl, 2 Caroline Schober, 1 Franz Dieber, 3 Josef Köfer, 3 

Rudolf Zechner, 1 and Ellen L. Zechner 1 

Institute of Molecular Biology, Biochemistry and Microbiology 1 and Institute of Hygiene, 2 Karl-Franzens University, 

and Department of Veterinary Administration in Styria, 3 Graz, Austria 

Received 4 November 2002/Returned for modification 8 January 2003/Accepted 4 March 2003 

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. 

* Corresponding author. Mailing address: Institute of Molecular 

Biology, Biochemistry and Microbiology, Karl-Franzens University, 

Graz, Heinrichstr. 31a, A-8010 Graz, Austria. Phone: 43-316-380-1902. 

Fax: 43-316-380-9016. E-mail: gregor.gorkiewicz@uni-graz.at. 

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). 

Species characterization. The identities of bacterial strains obtained from 

culture collections were verified by Gram staining and microscopy. Speciesspecific 

identification of field strains was performed by standard phenotypic tests, 

as described in the literature (26, 34, 36). Strains were initially analyzed for the 

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2537

2538 GORKIEWICZ ET AL. J. CLIN. MICROBIOL. 

TABLE 1. Sources of 16S rRNA sequences used in this study a 

Species Strain Source (if known) GenBank accession no. b Reference 

C. fetus subsp. fetus (n 15) ATCC 27374 T 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 



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 19438 T Bovine, England M65011 

V1-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 



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 



B99/222 Human, Austria (AF550623) This study 


B99/131 Human, Austria AF550625 This study 


ATCC 33559 T , CCUG 11283 c Porcine, Belgium M59073, L04312 

RMIT32A 

L19738 

C. jejuni subsp. jejuni (n 14) LMG 9217 Human, Belgium AF550626 This study 











ATCC 33560 T , CCUG 11284 c 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 

CCUG23947 T 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 

LMG7795 T 

X67775 

Continued on following page

VOL. 41, 2003 SPECIES-SPECIFIC IDENTIFICATION OF CAMPYLOBACTERS 2539 

TABLE 1—Continued 

Species Strain Source (if known) GenBank accession no. b Reference 

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 12470 T Feline U03022 

C. curvus (n 5) LMG 11034 Human, United States AF550650 This study 

LMG 11127 Belgium AF550651 This study 


ATCC 35224 T Human, United States L04313 

C10ETOH 

L06976 

C. concisus (n 5) LMG 7545 Human, Sweden AF550653 This study 

LMG 7961 Human, Sweden (AF550653) This study 


ATCC 33237 T Human L04322 

FDC 288 Tanner 

L06977 

C. showae (n 3) LMG 12636 Human, United States AF550655 This study 

CCUG 11641 Human, Sweden L06975 

CCUG 30254 T 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 33236 T Human, United States L04320 

L37787 

C. rectus (n 4) LMG 7611 Human, Sweden AF550659 This study 

LMG 7612 Human, Sweden (AF550659) This study 

ATCC 33238 T 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 6822 T Porcine L06978 

C. hyointestinalis subsp. 

hyointestinalis (n 10) 

C. hyointestinalis subsp. 

lawsonii (n 6) 


ATCC35217 Porcine, United States M65010 

NCTC11608 T 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 

CCUG 27631 Porcine, Sweden AF097683 19 

CHY 4 Porcine, England AF097684 19 





C. lanienae (n 5) S-K FAVW Porcine, Austria AF550664 This study 

NCTC 13004 T Human, Switzerland AF043425 30 

UB 994 Human, Switzerland AF043424 30 



C. hominis (n 4) NCTC 13146 T Human, England AJ251584 

HS-C Human, England AF062492 

HS-B Human, England AF062491 

HS-A Human, England AF062490 


a A total of 135 sequences were tested. 

b 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. 

c Synonymous strains derived from different culture collections.


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. 

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-TATGGAGAGTTTGATC 

CTGG-3) and Ps3/1 (5-GTTAAGCTGTTAGATTTCAC-3), Ps5/2 (5-AGC 

GTTACTCGGAATCACTG-3) and Ps3/2 (5-ACAGCCGTGCAGCACCTGT 

C-3), and Ps5/3 (5-AACCTTACCTGGGCTTGATA-3) and Ps3/3 (5-AAGG 

AGGTGATCCAGCCGCA-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-A 

CTTAACCCAACATCTCACG-3) were used to amplify a 334-bp DNA fragment 

containing the variable regions Vc5 and Vc6. Primers Vc1/2-F (5-AGAG 

TTTGATCCTGGCTCAG-3) and Vc1/2-R (5-TGATCATCCTCTCAGACCA 

G-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. 

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 g of ampicillin 

per ml, 20 g of 5-bromo-4-chloro-3-indolyl--D-galactopyranoside per ml, and 

0.1 mM isopropyl--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). 

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 amplifica- 

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tion 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, 

a 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. 

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 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 

Species 

% Variation a 

TABLE 2. Homology matrix 




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.


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/1D a 

C. helveticus 6D 5D 2D 1D 

C. curvus 6E 5C 2F 1B/1C b 

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 

a Strains LMG 11760 and CF89-12. 

b Strain C10ETHO. 

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). 

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. 

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). 


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. 



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). 

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