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

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-
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