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J Clin Microbiol. Dec 2004; 42(12): 5731–5738.
PMCID: PMC535295

Phylogenetic Analysis and PCR-Restriction Fragment Length Polymorphism Identification of Campylobacter Species Based on Partial groEL Gene Sequences

Abstract

The phylogeny of 12 Campylobacter species and reference strains of Arcobacter butzleri and Helicobacter pylori was studied based on partial 593-bp groEL gene sequences. The topology of the phylogenetic neighbor-joining tree based on the groEL gene was similar to that of the tree based on the 16S rRNA gene. However, groEL was found to provide a better resolution for Campylobacter species, with lower interspecies sequence similarities (range, 65 to 94%) compared with those for the 16S rRNA gene (range, 90 to 99%) and high intraspecies sequence similarities (range, 95 to 100%; average, 99%). A new universal reverse primer that amplifies a 517-bp fragment of the groEL gene was developed and used for PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of 68 strains representing 11 Campylobacter species as well as reference strains of A. butzlerii and H. pylori. Digestion with the AluI enzyme discriminated all Campylobacter species included in the study but showed more intraspecies diversity than digestion with the ApoI enzyme. A hippurate-negative variant of Campylobacter jejuni with a high level of groEL sequence similarity to both C. jejuni (96%) and C. coli (94%) gave a unique AluI profile and an ApoI profile identical to those of other C. jejuni strains. In conclusion, groEL gene sequencing and PCR-RFLP analysis are recommended as valuable tools for the identification of Campylobacter species.

Campylobacter jejuni is a major cause of bacterial gastroenteritis in humans (8). During the last two decades new Campylobacter species have been described, resulting in 16 species and 6 subspecies (5, 25). The clinical importance of some of these new species is as yet unknown (18). Present selective culture methods have been developed for the specific isolation of C. jejuni and other thermotolerant Campylobacter species (3). The prevalence of the more fastidious species in human disease and the environment may thus be underestimated. The asaccharolytic nature and inertness of Campylobacter and related species in traditional biochemical tests makes their identification difficult. In addition, the identification of Campylobacter species according to phenotypic properties may result in false identifications, as many species include strains that give atypical results in some key phenotypic tests (24, 28). For example, C. jejuni strains that lack the ability to hydrolyze hippurate have been described (37). To further understand the epidemiology and impact of Campylobacter species on public and animal health, better isolation and identification methods are required.

Various molecular DNA-based methods for the identification of Campylobacter species have been developed. These methods typically require the use of several species-specific PCR primers, hybridization probes, or multiple restriction enzymes and are usually not designed to differentiate all known species simultaneously. 16S rRNA gene sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis have been described for Campylobacter species identification (2, 9, 22), but these methods do not differentiate between C. jejuni and C. coli. PCR-RFLP analysis of the 23S rRNA gene with two restriction enzymes is able to discriminate between Campylobacter species, but interpretation of the results is complicated by intervening sequences (11). More recently, amplified fragment length polymorphism fingerprinting has proven to be useful for Campylobacter species identification (4, 27), but the method is laborious and expensive. Whole-genome DNA-DNA hybridization analysis allows species identification, but the method is not suitable for routine use (34).

The groEL gene, which encodes a 60-kDa subunit (known as GroEL, 60-kDa chaperonin, and heat shock protein 60) of a complex that assists with the three-dimensional folding of bacterial proteins (7), has the potential to serve as a general phylogenetic marker because of its ubiquity and conservation in nature (32). Studies on the suitability of a fragment from a conserved region of the groEL gene for phylogenetic analyses and identification of species of the genera Bifidobacterium (13), Helicobacter (23), Rickettsia (20), Staphylococcus and Macrococcus (16), and Vibrio (17), among others, have been published. These studies have shown that, despite the conserved nature of the groEL gene, the level of interspecies groEL sequence variation is greater than that of the 16S rRNA gene, providing better resolution for species classification. Recently, partial groEL sequences from reference strains of C. jejuni, C. coli, and C. lari have produced similar results (39). Dot blot hybridization and PCR-RFLP analysis with AluI enzyme digestion of the partial groEL gene amplicon were also evaluated for the identification of C. jejuni, C. coli, and C. fetus subsp. intestinalis, with species-specific results (39).

In the present study, we cloned and sequenced 593 bp of the groEL gene from strains representing 11 Campylobacter species and a reference strain of Arcobacter butzleri. A new degenerate reverse primer applicable for direct sequencing was designed for specific amplification of 517 bp of the gene. PCR-RFLPs with AluI and ApoI digestion of the partial groEL gene amplicon were assessed by using previously characterized human and animal Campylobacter isolates.

MATERIALS AND METHODS

Bacterial strains.

The bacterial strains used in this study are listed in Table Table1.1. Campylobacter strains isolated in Finland were characterized by microscopy (Gram stain, motility); for catalase and oxidase activity, hippurate and indoxyl acetate hydrolysis, nitrate reduction, and H2S production in triple sugar iron agar; and with the API Campy system (bioMérieux sa, Marcy l'Etoile, France), as appropriate. C. helveticus and C. upsaliensis isolates were identified by species-specific PCR (19).

TABLE 1.
Sources, PCR-RFLP profiles, and sequence accession numbers of the strains included in the study

Human isolate 6871, tentatively identified as C. coli, was further characterized by hippuricase PCR (21); 16S rRNA gene sequencing with primers 27f, 518r, 536f, 1054r, 1073f, and 1492r (10, 26); 23S rRNA gene PCR-RFLP analysis (11); and dot blot hybridization (12). Hybridization was performed with genomic DNA isolated from C. jejuni reference strain NCTC 11168 and labeled with digoxigenin-11-dUTP (DIG-High Prime; Roche Diagnostics GmbH, Mannheim, Germany) as the probe. Genomic DNAs from C. coli CCUG 11283 and C. jejuni NCTC 11168 were used as references for evaluation of the degree of hybridization. The hybridizations were carried out at 58°C.

Isolation of genomic DNA.

Genomic DNA was isolated from cultures grown on brucella blood agar for 24 to 48 h at 37°C under microaerobic conditions, as described previously (12, 29). Alternatively, for testing of the rapid PCR-RFLP method, cell lysates were prepared by suspending a 10-μl loopful of growth in 500 μl of sterile distilled water in a microcentrifuge tube. The tubes were heated at 100°C for 10 min and subsequently cooled to 4°C. The tubes were centrifuged at 13,000 rpm (Biofuge 13, rotor 3757; Heraeus Sepatech GmbH, Osterode/Harz, Germany) for 5 min, and the supernatant was stored at −20°C.

PCR amplification and cloning of partial groEL gene.

The partial (593-bp) groEL gene was amplified with the degenerate primers H60F (5′-GGN GAY GGN CAN CAN CAN GCN CAN GT-3′) and H60R (5′-TCN CCR AAN CCN GGN GCY TTN CAN GC-3′) (30) (see below for explanation of bases designated Y, N, D, or R). The amplified region corresponds to nucleotides 253 to 845 of the groEL gene in C. jejuni NCTC 11168 (GenBank accession no. AY044099). The 50-μl PCR mixture contained 200 ng of genomic DNA (or 5 μl of cell lysate), 100 μM each deoxynucleoside triphosphate, 2.5 U of Taq DNA polymerase (MBI Fermentas, Hanover, Md.), 10 mM Tris-HCl (pH 8.8 at 25°C), 50 mM KCl, 0.08% Nonidet P-40, 1.5 mM MgCl2, and 2 μM each primer. The PCR thermal cycling conditions were as described earlier (13). PCR products of the expected sizes were purified from 2% NuSieve GTG low-melting-point agarose gels (BioWhittaker Molecular Applications, Rockland, Maine) with a QIAquick gel extraction kit (Qiagen GmbH, Hilden, Germany), cloned into pGEM-T Easy vector (Promega, Madison, Wis.), and transformed into competent JM109 Escherichia coli cells (Promega). The transformants were selected with ampicillin, and recombinants were selected by blue-white differentiation. Plasmids were isolated from several clones with a Qiagen Plasmid Mini kit. To check for the presence of the correct 593-bp insert, plasmids were digested with EcoRI and the restriction products were separated on 1% agarose gels.

DNA sequencing.

Nucleotide sequencing of three clones per strain was performed by automated cycle sequencing with Big Dye terminators (ABI 377XL; PE Applied Biosystems, Foster City, Calif.) and primers RP (reverse primer) and UP (universal primer M13-20).

Sequence analysis.

The partial groEL gene sequences determined in this study and those available in public databases (Table (Table1)1) were analyzed with Bionumerics software (version 3.5; Applied Maths BVBA, Sint-Martens-Latem, Belgium). Distances were calculated by using all 541 bases (with the omission primer sequences). After pairwise and multiple-sequence alignments, a phylogenetic tree was constructed by the neighbor-joining method (31). Distances were corrected for multiple base changes by the method of Jukes and Cantor (14). The topology of the tree was evaluated by 1,000 trials of bootstrap analysis.

The groEL nucleotide sequences were translated to the corresponding amino acid sequences with the Transeq program (EMBOSS [European Molecular Biology Open Software Suite]). The resulting sequences were aligned, and a neighbor-joining tree was calculated with the ClustalW program. The phylogenetic tree based on the amino acid sequences was drawn with the TreeView program (version 1.6.6; Division of Environmental and Evolutionary Biology, University of Glasgow [http://taxonomy.zoology.gla.ac.uk/rod/rod.html]).

Design of new reverse PCR primer.

A new degenerate reverse PCR primer was developed for the conserved protein sequence AEDIEGE. The nucleotide sequence of the new reverse primer H60R1 was 5′-C YTC NCC YTC DAT RTC YTC NGC-3′ (517-bp product, corresponding to nucleotides 253 to 769 of the groEL gene in C. jejuni NCTC 11168, where Y is C or T; N is G, A, T, or C; D is G, T, or A; and R is A or G). The new primer was developed to have a melting temperature closer to that of the forward primer and to increase the specificity of the PCR. The melting temperature, primer-dimer, and secondary structure formation were checked by using the Sigma-Genosys basic oligonucleotide calculator (http://www.sigma-genosys.co.uk/oligos/frameset.html).

PCR-RFLP typing of Campylobacter isolates.

On the basis of computational restriction fragment analysis of the partial groEL gene sequences with the Restriction Mapper program (version 3; http://www.restrictionmapper.org/), the AluI and ApoI restriction enzymes were chosen, as they were expected to yield sufficient fragment numbers of sufficient sizes to produce species-specific restriction profiles. Digestions were performed with 20 μl of the PCR products in a total volume of 25 μl with 5 U of AluI or 4 U of ApoI (New England Biolabs Inc., Beverly, Mass.). The resulting fragments were separated electrophoretically (90 V for 3 h) in 4% MetaPhor agarose gels in 1× Tris-acetate-EDTA buffer. The gels were stained with ethidium bromide and visualized under UV light. The resulting patterns were analyzed with BioNumerics software.

Nucleotide sequence accession numbers.

The partial groEL and 16S rRNA gene sequences obtained in this study were deposited in GenBank. The accession numbers are shown in Table Table1.1. For comparison, published groEL and 16S rRNA gene sequences were downloaded from GenBank (Table (Table11).

RESULTS

Phylogenetic analysis of the partial groEL gene sequences compared with 16S rRNA gene sequences.

The neighbor-joining tree constructed from the partial groEL gene sequences is shown in Fig. Fig.1.1. The major topology of the tree based on the partial groEL gene sequences was similar to that based on the 16S rRNA gene sequences. The more detailed similarity analysis of the partial groEL and 16S rRNA gene sequences among Campylobacter species, A. butzleri, and Helicobacter pylori is shown in Table Table2.2. The similarities of the groEL sequence of Campylobacter species to those of A. butzleri strain CCUG 10373 and H. pylori strain 26695 were in the range of 51 to 73% and 56 to 67%, respectively, whereas the 16S rRNA gene sequence similarities were 83 to 85% and 81 to 83%, respectively (Table (Table2).2). The interspecies groEL sequence similarities between Campylobacter species ranged from 65% (between C. fetus subsp. fetus and C. rectus) to 94% (between C. jejuni and C. coli) and 100% (between C. hyoilei and C. coli) (Fig. (Fig.1).1). C. upsaliensis and C. helveticus formed a separate branch with high bootstrap support, as did C. coli and C. jejuni as well as C. lanienae, C. fetus subsp. fetus, and both C. hyointestinalis subspecies (Fig. (Fig.1).1). The partial groEL gene sequences of the type strains of C. upsaliensis and C. helveticus showed 91% similarity, whereas the 16S rRNA gene sequences showed 98% similarity (Table (Table2).2). The 16S rRNA gene sequence similarity between C. helveticus and C. jejuni was high (97%), but the partial groEL gene similarity was only 84%. The groEL sequence similarity of C. coli and C. jejuni was 91%, whereas the 16S rRNA gene sequence similarity was 98%. C. lari showed 86 and 87% groEL gene sequence similarity to C. jejuni and C. coli, respectively, whereas the 16S rRNA gene sequence similarities were 98 and 97%, respectively. Similarly, the similarity of the C. lanienae groEL gene sequence to those of C. hyointestinalis subsp. hyointestinalis and C. hyointestinalis subsp. lawsonii was 81%, whereas the 16S rRNA gene sequence similarities were 97 and 98%, respectively. The groEL sequence similarity between C. fetus and C. hyointestinalis subsp. hyointestinalis and C. hyointestinalis subsp. lawsonii was 85%.

FIG. 1.
Neighbor-joining tree based on partial 541-bp groEL sequences. The tree was rooted with H. pylori. The number at each branch point represents the percentage of bootstrap support calculated from 1,000 trees. The scale bar represents the sequence divergence. ...
TABLE 2.
Similarity analysis of partial groEL and 16S rRNA sequences among Campylobacter species, A. butzleri, and H. pylori

Strain 6871 had a higher level of groEL sequence similarity to the C. jejuni strains (94.6 to 96%) than to the C. coli strains (92.6 to 93.6%). The strain was negative by the hippurate hydrolysis test and hippuricase gene PCR. The partial (1,470-bp) 16S rRNA gene sequence of strain 6871 showed the highest degree of similarity to C. jejuni strains ATCC 43431 (99.9%) and NCTC 11168 (99.7%) in a search with the BLAST algorithm. The level of sequence similarity to the type strain of C. coli CCUG 11283 was, however, also high (98.0%). PCR-RFLP analysis of the 23S rRNA gene resulted in a C. jejuni-specific profile for strain 6871. Dot blot hybridization of the whole genomic DNA of strain 6871 gave a more intense signal with C. jejuni than with the C. coli reference strain.

The intraspecies sequence similarities for C. coli ranged from 99 to 100%, those for C. jejuni ranged from 95% (98% when the hippurate-negative C. jejuni strain 6871 was excluded) to 100%, and those for C. lari ranged from 99 to 100%. The intraspecies sequence similarities between the two strains of C. helveticus and C. upsaliensis were 99.4 and 97.6%. The sequence similarity between C. hyointestinalis subsp. hyointestinalis and C. hyointestinalis subsp. lawsonii was 97.9%. The sequence similarity between C. sputorum biovar sputorum and C. sputorum subsp. bubulus was 99.8%.

The neighbor-joining tree based on the deduced partial GroEL amino acid sequences (180 amino acids) is shown in Fig. Fig.2.2. Hippurate-negative C. jejuni strain 6871 had 100% amino acid similarity to C. jejuni strain 4117. The amino acid similarities between C. upsaliensis (two strains) and C. hyointestinalis subsp. hyointestinalis and C. hyointestinalis subsp. lawsonii were 100%. The intraspecies amino acid similarities of the other species were equal to the nucleotide sequence similarities, whereas the interspecies amino acid similarities were higher than the nucleotide sequence similarities, ranging from 84% (between C. hyointestinalis and C. lari) to 99% (between C. jejuni and C. coli) and 100% (between C. hyoilei and C. coli). A. butzleri strain CCUG 10373 and H. pylori strain 26695 had GroEL amino acid sequence similarities to the Campylobacter species GroEL amino acid sequences that ranged from 70 to 75% and 77 to 81%, respectively.

FIG. 2.
Neighbor-joining tree based on the deduced partial 180-amino-acid GroEL sequences. The tree was rooted with H. pylori. The scale bar represents the sequence divergence.

PCR-RFLP identification of Campylobacter species based on partial 517-bp groEL gene amplicons.

The results of the computational restriction fragment length analysis with the AluI and ApoI enzymes are shown in Table Table3.3. According to these results, all Campylobacter species studied had species-specific AluI digestion patterns and all but C. helveticus and C. mucosalis and C. rectus and H. pylori had species-specific ApoI digestion patterns. The AluI and ApoI digestion patterns of the 517-bp groEL gene amplicons of representative Campylobacter strains from 11 species and 3 subspecies or biovars, as well as reference strains of A. butzleri and H. pylori, are shown in Fig. Fig.3.3. Table Table11 lists the profiles obtained for the remaining strains.

FIG. 3.
RFLP analysis of the partial 517-bp groEL gene amplicon digested with AluI and ApoI. The combined dendrogram was derived with BioNumerics software by using the unweighted pair group method with arithmetic averages and the Dice coefficient with 1% optimization ...
TABLE 3.
Computational restriction fragment length analysis of the partial groEL gene product obtained with the AluI and ApoI enzymesa

Visually, 19 and 10 separate profiles were obtained by AluI and ApoI digestion, respectively. AluI digests alone discriminated among all species included in the study. For C. coli, C. jejuni, C. lari, and C. upsaliensis, three, two, two, and four separate AluI restriction profiles, respectively, were observed. Hippurate-negative strain C. jejuni 6871 was the only strain to yield AluI restriction profile 8b. Two ApoI restriction patterns were observed for C. coli. The ApoI restriction patterns could not differentiate between C. helveticus and C. mucosalis, C. hyointestinalis and C. fetus, or the less common ApoI profile 6 of C. coli (observed in 6 of 21 cases) and C. jejuni.

An atypical restriction profile for C. coli isolate S161R from a pig was observed. After the subculture of separate colonies, this isolate was subsequently identified as a coculture of two C. coli strains, strains S161Ra and S161Rb, representing two separate AluI-ApoI profiles, 10a-8 and 10c-6, respectively.

DISCUSSION

The major topology of the phylogenetic neighbor-joining tree constructed from the partial groEL gene sequences was similar to that constructed from the 16S rRNA gene sequences. However, groEL was found to provide better resolution for Campylobacter species, with lower interspecies sequence similarities (range, 65 to 94%) compared to that obtained with the 16S rRNA gene (range, 90 to 99%). The intraspecies groEL gene sequence similarities (range, 95 to 100%; average, 99%) were high compared to the interspecies sequence similarities, and strains representing a distinct Campylobacter species or two subspecies clustered tightly together. These results suggest an added value of the groEL gene sequence over the 16S rRNA gene sequence for Campylobacter species identification.

The most important phenotypic test used to differentiate between C. jejuni and C. coli is hydrolysis of hippurate, which relies on the activity of the hippuricase enzyme characteristic for C. jejuni. Strain 6871 was identified as an atypical (hippurate-negative) C. jejuni strain, according to PCR-RFLP analysis of the 23S rRNA gene and dot blot hybridization. Similar hippurate-negative C. jejuni strains have been shown to be quite uncommon (33, 37). In one study, 1.6% of human and poultry strains were reported to be hippurate negative according to the results of quantitative DNA hybridizations and hippurate hydrolysis experiments (37). In another study, some C. jejuni strains (less than 1%) did not produce a PCR product for the hippuricase gene (33). Hippurate-negative strains producing a hippuricase PCR product smaller than expected have also been reported (6, 35). Strain 6871 had a unique AluI profile (profile 8b), whereas all other C. jejuni strains showed identical profiles. Strain 6871 also showed only 94.6 to 96% groEL sequence similarity with C. jejuni, whereas the intraspecies sequence similarities among the other strains ranged from 98 to 100%. Hippurate-negative C. jejuni strains may represent a distinct clonal lineage of C. jejuni, as proposed previously (15).

Strains first described as a distinct species, C. hyoilei (1), have subsequently been proposed to represent a variant of C. coli, according to indistinguishable results by 66 phenotypic tests and a high DNA-DNA hybridization level (38). The type strains of these two species showed 100% groEL sequence similarity. C. sputorum subsp. bubulus has been reclassified as C. sputorum biovar sputorum (26). The two type strains of C. sputorum studied had a high groEL sequence similarity (99.8%), supporting earlier findings.

The new degenerate reverse primer H60R1 yielded specific amplification of the desired 517-bp fragment of the groEL gene and was used as the basis for the PCR-RFLP assay. It was also successfully tested for use for the direct sequencing of the partial groEL gene. With direct sequencing the cumbersome cloning step may be omitted, and sequence data for novel Campylobacter species and/or isolates can easily be obtained and evaluated against known species. Because the sequence divergence of the groEL gene is much higher, only one primer pair is required for species identification, whereas three primer pairs (or four primer pairs for specific variable regions) are required for 16S rRNA gene sequencing (9). Another advantage of the partial groEL gene is that it has not been shown to contain intervening sequences, which complicate the alignment and comparison of 16S and 23S rRNA gene sequences and the interpretation of the results of PCR and PCR-RFLP assays based on these genes (25).

AluI PCR-RFLP analysis was found to give species-specific discrimination. In contrast to an earlier report of a study that used a limited number of C. coli, C. jejuni, and C. fetus subsp. intestinalis strains (39), more than one AluI profile was seen for the C. coli, C. jejuni, C. lari, and C. upsaliensis strains tested in our study. The profiles of a single species differed by two to three fragments. Due to genetic drift, which results in new PCR-RFLP profiles, the use of two restriction enzymes may be recommended. ApoI produced two profiles only for C. coli strains, one of which was identical to C. jejuni profile 6. ApoI did not discriminate between C. helveticus and C. mucosalis, C. rectus and H. pylori, or C. hyointestinalis and C. fetus; yet all of these species could be easily differentiated by their AluI profiles. In contrast, AluI profile 7a of C. lari was highly similar to profile 8a of C. jejuni, but these species could be distinguished by use of computer-assisted analysis and the respective ApoI profiles. Thus, only one or two restriction enzymes are needed to identify Campylobacter isolates to the species level, whereas two (22) and six (2) restriction enzymes are needed for the 16S rRNA gene-based PCR-RFLP methods, which additionally lack the ability to differentiate between C. jejuni and C. coli.

In conclusion, our results show that partial groEL gene sequencing and PCR-RFLP analysis are more suitable and simple methods for Campylobacter species-specific identification than the respective analyses of the 16S rRNA gene. Taxonomic studies of novel Campylobacter species are likely to benefit from the groEL gene sequence information. Further studies are needed to confirm the numbers of species-specific AluI and ApoI profiles and the utility of the PCR-RFLP assay.

Acknowledgments

This work was supported by grants from the Helsinki University Research Funds and the Academy of Finland.

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