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

VOL. 41, 2003 SPECIES-SPECIFIC IDENTIFICATION OF CAMPYLOBACTERS 2545
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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