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

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2540 GORKIEWICZ ET AL. J. CLIN. MICROBIOL. 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- Downloaded from jcm.asm.org at UNIVERSITATSBIBLIOTHEK on June 1, 2010
VOL. 41, 2003 SPECIES-SPECIFIC IDENTIFICATION OF CAMPYLOBACTERS 2541 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 Downloaded from jcm.asm.org at UNIVERSITATSBIBLIOTHEK on June 1, 2010
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