J Med Microbiol 55 (2006), 393-399; DOI: 10.1099/jmm.0.46282-0
© 2006 Society for General Microbiology
ISSN 0022-2615
Identification of Campylobacter spp. and discrimination from Helicobacter and Arcobacter spp. by direct sequencing of PCR-amplified cpn60 sequences and comparison to cpnDB, a chaperonin reference sequence database
Janet E. Hill1,
,
Ana Paccagnella2,
Kee Law2,
Pasquale L. Melito3,
David L. Woodward3,
Lawrence Price3,
Amy H. Leung4,
Lai-King Ng3,
Sean M. Hemmingsen1 and
Swee Han Goh2,4,5
1 National Research Council Plant Biotechnology Institute, Saskatoon, Saskatchewan, Canada
2 British Columbia Centre for Disease Control, 655W 12th Ave, Vancouver, BC, V5Z 4R4, Canada
3 Bacteriology and Enteric Diseases Program, National Microbiology Laboratory, Winnipeg, Manitoba, Canada
4 ,5 Dept of Pathology and Laboratory Medicine4 , and UBC CDC5 , University of British Columbia, BC, Canada
Correspondence
Swee Han Goh
shgoh{at}interchange.ubc.ca
Received 4 August 2005
Accepted 6 December 2005
A robust method for the identification of Campylobacter spp. based on direct sequencing of PCR-amplified partial cpn60 sequences and comparison of these to a reference database of cpn60 sequences is reported. A total of 53 Campylobacter isolates, representing 15 species, were identified and distinguished from phenotypically similar Helicobacter and Arcobacter strains. Pairwise cpn60 sequence identities between Campylobacter spp. ranged from 71 to 92 %, with most between 71 and 79 %, making discrimination of these species obvious. The method described overcomes limitations of existing PCR-based methods, which require time-consuming and complex post-amplification steps such as the cloning of amplification products. The results of this study demonstrate the potential for use of the reference chaperonin sequence database, cpnDB, as a tool for identification of bacterial isolates based on cpn60 sequences amplified with universal primers.
Abbreviations: BCCDC, British Columbia Centre for Disease Control; NML, National Microbiology Laboratory.
Present address: Dept of Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK, Canada. 
The GenBank/EMBL/DDBJ accession numbers for the cpn60 sequences determined in this study are DQ059425–DQ059481.
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INTRODUCTION
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Although >95 % of human Campylobacter infections are caused by Campylobacter jejuni and Campylobacter coli, numerous other species of Campylobacter, including Campylobacter upsaliensis, C. jejuni subsp. doylei, Campylobacter fetus subsp. fetus, Campylobacter concisus, Campylobacter lari, Campylobacter sputorum, Campylobacter mucosalis, Campylobacter rectus and Campylobacter hyointestinalis, have been shown to cause disease in humans (Butzler, 2004). Twenty-five species of Campylobacter from human and other animal sources have been defined (Euzéby, 1997), although the clinical significance of some species is not known, and identification and discrimination of clinically significant strains remains challenging. A panel of phenotypic tests have been used for Campylobacter species identification based on biochemical profiling (On, 1996), but interpretation of the results can be problematic due to inconsistencies in phenotype within species. For example, an important tool for the discrimination of C. coli and C. jejuni is the hippurate hydrolysis assay in which C. jejuni isolates are hippurate-positive; however, hippurate-negative C. jejuni strains have been observed (Totten et al., 1987), and different assay methods have been found to give variable results (Morris et al., 1985). Identification of non-C. jejuni/C. coli Campylobacter species by biochemical profiling is also challenging due to the inconsistency of the phenotypic profiles observed among strains within these species (On, 1996). Thus, robust molecular tests are required for differentiating between various Campylobacter species, especially for those known to commonly cause disease in humans and animals (Nachamkin, 2003). Identification is further complicated by the similar phenotypic and biochemical profiles of other related Gram-negative curved rods, Arcobacter and Helicobacter.
Various DNA-based methods have been developed, including those based on analysis of sequences or RFLPs of amplified rRNA sequences, amplified fragment length polymorphism fingerprinting or the application of species-specific PCR methods for the detection of individual species (Cardarelli-Leite et al., 1996; Gorkiewicz et al., 2003; Marshall et al., 1999; Duim et al., 2001; On & Harrington, 2000). Unfortunately, these methods are limited by their complexity or their inability to distinguish between closely related species such as C. coli and C. jejuni. Methods based on amplification or hybridization using targets such as glyA, ceuE or a putative GTPase-encoding gene (Van Doorn et al., 1999; Al Rashid et al., 2000; Gonzalez et al., 1997) showed some promise, but the demonstrated applications showed discrimination amongst only a limited range of Campylobacter species, and studies often did not include potentially confounding Arcobacter and Helicobacter spp.
More recently, gene targets providing more discriminating information, such as omp50 and cpn60 (also known as the 60 kDa chaperonin, groEL or hsp60), have been found to provide the necessary inter-species discrimination within the genus Campylobacter (Dedieu et al., 2004; Kärenlampi et al., 2004). However, the omp50 target was found to be absent from over 90 % of C. coli strains, requiring the application of additional methods for identification of these strains (Dedieu et al., 2004). While offering excellent discriminating power, the cpn60-based method described by Kärenlampi et al. (2004) required RFLP analysis and/or cloning of PCR products prior to sequencing, adding time, cost and complexity, and limiting the utility of the method in a clinical laboratory.
The cpn60 gene encodes the 60 kDa chaperonin protein that is found in virtually all eubacteria, some Archaea, and in the plastids and mitochondria of eukaryotes. The utility of this target for bacterial species identification, detection, quantification and phylogenetic analysis, and for complex microbial community profiling has been well established (Dumonceaux et al., 2006; Eriks et al., 1996; Goh et al., 1997; Hill et al., 2002, 2005; Jian et al., 2001; Zeaiter et al., 2002). Its use as a target for identification of bacterial isolates in a clinical laboratory setting is facilitated by the availability of degenerate, universal PCR primers for the amplification of the approximately 555 bp region of cpn60 corresponding to nucleotides 274–828 of the Escherichia coli cpn60 sequence (Goh et al., 1996). More recently, a reference database of cpn60 sequences, cpnDB, has been made freely available via a web interface (Hill et al., 2004), permitting users to compare the cpn60 sequence of an isolate to thousands of type strain, reference strain and clinical isolate sequences. Here we demonstrate how this resource can be exploited in the identification of Campylobacter spp. and their distinction from phenotypically similar Helicobacter and Arcobacter spp.
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METHODS
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Bacterial strains.
Strains used in this study are described in Table 1
. Type strains were obtained from the American Type Culture Collection. Campylobacter strains provided by the National Microbiology Laboratory (NML) were cultured and identified to the species level using established procedures for biotyping (On, 1996), fatty acid profiling (Lambert et al., 1987), 16S rRNA PCR–RFLP (Marshall et al., 1999) and 16S rRNA gene sequencing (Edwards et al., 1989). Clinical isolates from the British Columbia Centre for Disease Control (BCCDC) were identified to the genus level using standard phenotypic methods: evaluation of oxidase and catalase activity, resistance to nalidixic acid, resistance to cephalothin, hippurate hydrolysis and indoxyl acetate hydrolysis (Mills & Gherna, 1987; Morris et al., 1985; Nachamkin, 2003; Versalovic & Fox, 2003).
Genomic DNA extraction and cpn60 PCR.
Extraction of crude DNA from bacterial cultures by the Instagene method (Bio-Rad) was performed according to the manufacturer's protocol. PCR reactions contained 5 µl Instagene extract, 2·5 U Taq DNA polymerase (Invitrogen), 2·5 mM MgCl2, 50 mM KCl, 10 mM Tris/HCl pH 8·3, 250 µM each of the dNTPs and 20 pmol each of degenerate primers H729 (5'-CGC CAG GGT TTT CCC AGT CAC GAC GAI III GCI GGI GAY GGI ACI ACI AC-3') and H730 (5'-AGC GGA TAA CAA TTT CAC ACA GGA YKI YKI TCI CCR AAI CCI GGI GCY TT-3'), which include landing sites for sequencing primers M13(–40)F and M13(48)R (underlined). PCR reactions were conducted in two different laboratories, using different PCR machines. Temperature cycling was performed on a Bio-Rad iCycler (95 °C for 2 min, followed by 40 cycles of 30 s at 95 °C, 30 s at 46 °C, 30 s at 72 °C, and a final extension at 72 °C for 5 min) or a Stratagene Robocycler (95 °C for 3 min, followed by 40 cycles of 1 min at 94 °C, 2 min at 37 °C and 5 min at 72 °C, and a final extension at 72 °C for 10 min). Optimal PCR parameters for a given amplification will vary with the thermocycler used (due to variations in factors such as sample temperature ramp time). Both of the protocols described here efficiently produced PCR product for sequencing. PCR products were purified using the QIAquick Spin PCR purification kit (Qiagen) and sequenced directly using primers M13 (–40) F and M13 (48) R. The 555 bp cpn60 sequences of clinical isolates were compared to cpnDB (http://cpndb.cbr.nrc.ca) using FASTA (Hill et al., 2004; Pearson & Lipman, 1988). Phylogenetic analysis was conducted using the PHYLIP software package (Felsenstein, 1989).
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RESULTS AND DISCUSSION
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Sequences of cpn60 for each of the strains listed in Table 1
were determined and have been deposited in GenBank with the accession numbers listed. The primers employed for PCR amplification of cpn60 sequences include landing sites for standard sequencing primers, permitting the direct sequencing of PCR products and the elimination of the time-consuming cloning steps required by other methods for Campylobacter identification using this gene target (Kärenlampi et al., 2004). Direct sequencing also virtually eliminates the impact of Taq DNA polymerase-induced sequence artefacts that confound methods based on cloned PCR products where each clone originates from a single, potentially error-containing PCR amplicon.
The phylogenetic relationships of the reference and clinical strains of Campylobacter, Helicobacter and Arcobacter are illustrated in Fig. 1. The Campylobacter species were clearly distinguished from each other, and from the Arcobacter and Helicobacter type and reference species examined. In addition, the NML clinical isolates were found to cluster with reference strains, in accordance with their initial identification.
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Fig. 1. Inferred phylogenetic relationships of Campylobacter, Helicobacter and Arcobacter strains based on partial cpn60 sequences. The tree is a consensus of 500 neighbour-joined trees. Bootstrap values (out of 500) are indicated at branch-points. The sources of the strains are described in Table 1.
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Pairwise DNA identities between the 15 species of Campylobacter included in this study ranged from 71 to 92 % (excluding the identical pair of C. coli and Campylobacter hyoilei discussed below). Of the 105 pairwise identities, 78 were between 71 and 79 %, and 23 were between 80 and 89 %. Only C. upsaliensis and Campylobacter helveticus (92 % identical), and C. coli and C. jejuni (90 % identical) had identities 
90 %. For species clusters containing multiple strains or isolates, with the exception of C. coli, the intra-species pairwise DNA identities were uniformly high: 96–100 % (C. upsaliensis), 95–98 % (C. lari), 97–100 % (C. jejuni), 100 % (C. hyointestinalis) and 99–100 % (C. fetus).
The eight reference strains of C. coli, including the type strain ATCC33559T, were found to form a relatively broad cluster with pairwise sequence identities ranging from 94–100 %, and the type strain of C. hyoilei (ATCC51729T) was found to be identical to the C. coli type strain (ATCC33559T) as reported previously (Kärenlampi et al., 2004). Two distinct groups were evident within the C. coli cluster (Fig. 1). One group contains 13 strains, including the type strains of C. hyoilei and C. coli, and has inter-strain sequence identities of 96–100 %. Another separate and distinct group encompasses six C. coli reference and clinical strains with pairwise sequence identities of 99–100 %, and may represent the typical C. coli taxon. Sequence identities between the two C. coli groups are 94–96 %.
C. hyoilei was originally described by Alderton et al. (1995) and strains of this organism were implicated in causing proliferative enteritis in pigs. Vandamme et al. (1997) provided evidence that C. hyoilei (Alderton et al., 1995) and C. coli (Véron & Chatelain, 1973) were subjective synonyms and it has been suggested that C. hyoilei is a variant of C. coli specialized for the porcine gastrointestinal tract (Vandamme & On, 2001). However, Dep et al. (2001) have provided molecular and phenotypic evidence that C. hyoilei and C. coli type strains are distinct Campylobacter species. Using hybridization methods, Metherell et al. (1999) found that the C. coli type strain, NCTC 11366 (ATCC33559T), was atypical for C. coli. Our data, based on the cpn60 sequences from the type strains, other ATCC C. coli reference strains and clinical isolates, suggest that C. hyoilei may indeed be a valid species since the C. coli type strain is identical in sequence to it and is considered an atypical C. coli. Interestingly, human and animal isolates are found in both clusters.
The strain identification method demonstrated here offers time and cost advantages over previously described DNA-based methods for identification and discrimination of Campylobacter species (Cardarelli-Leite et al., 1996; Dedieu et al., 2004; Duim et al., 2001; Gorkiewicz et al., 2003; Kärenlampi et al., 2004; Marshall et al., 1999; On & Harrington, 2000) in that it involves direct sequencing of PCR-amplified cpn60 sequences with no requirement for the cloning of PCR products, a method that could easily be adapted to a high-throughput mode. We have applied the universal, degenerate primers described here to the amplification and sequencing of hundreds of reference and type strain organisms, including human and animal pathogens (Brousseau et al., 2001; Goh et al., 1997, 1998, 2000), incorporating this data into the cpnDB database of cpn60 sequences (Hill et al., 2004). This data is intensely curated, containing only complete, unambiguous cpn60 target sequences (amplified by primers H729 and H730) from identified sources (reference strains, type strains, clinical isolates). The database webpage provides users with free, on-line analysis tools for sequence identification, and has greatly enhanced the functionality of this diagnostic method, facilitating its application in phylogenetic studies, and the identification of established and emerging pathogens.
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ACKNOWLEDGEMENTS
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The authors gratefully acknowledge Jennifer Town for excellent technical assistance.
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TOP
INTRODUCTION
METHODS
