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

VOL. 41, 2003 SPECIES-SPECIFIC IDENTIFICATION OF CAMPYLOBACTERS 2543
TABLE 3. Pattern distribution among Campylobacter species
Species
Variable region
Vc6 Vc5 Vc2 Vc1
C. fetus 6A 5A 2A 1A
C. hyointestinalis 6B/6D 5A/5B/5C 2A/2B/2C 1A/1B
C. lanienae 6D 5B/5C 2C 1B
C. mucosalis 6C 5C 2C 1C
C. upsaliensis 6C 5D 2D 1D
C. coli 6D 5D/5B 2E 1D
C. jejuni 6D 5D 2E 1D
C. lari 6D 5D 2E 1F/1D a
C. helveticus 6D 5D 2D 1D
C. curvus 6E 5C 2F 1B/1C b
C. sputorum 6E 5E 2G 1G
C. concisus 6E 5C 2H 1B
C. rectus 6E 5F 2I 1C
C. showae 6E 5F 2J 1C
C. gracilis 6E 5E 2K 1E
C. hominis 6E 5G 2L 1H
a Strains LMG 11760 and CF89-12.
b Strain C10ETHO.
and strain LMG 11760 was a nalidixic acid-susceptible C. lari
strain. Their minimal sequence diversities from the sequences
of C. coli (0.5%) and C. jejuni (0.6%) were significantly different
from those of the classical C. lari strains compared to the
sequences of C. coli and C. jejuni (1.6%) (P 0.001). The 16S
rDNA sequence diversities among Campylobacter species are
given in Table 2.
Characterization of variable regions within the 16S rDNA.
To improve the analysis, we investigated whether particular
regions of 16S rDNA yield sufficient information to discriminate
among the taxa. 16S rDNA alignment studies revealed
four variable gene regions, which were termed Vc6, Vc5, Vc2,
and Vc1, in accordance with the variable regions of the procaryotic
16S rRNA. These regions displayed a high level of
interspecies sequence variation. Among these we discerned
several sequence patterns that are applicable for species-specific
identification. Figures 3A to D show the alignments of the
Campylobacter 16S rDNA sequences corresponding to the Vc
regions. Campylobacter species were grouped according to the
particular sequence patterns within the respective Vc regions.
Five distinct patterns, termed 6A to 6E, were found in the Vc6
region (Fig. 3A). Seven patterns, termed 5A to 5G, were defined
in Vc5 (Fig. 3B). Twelve patterns, termed 2A to 2L, were
defined in Vc2 (Fig. 3C). Analysis of Vc1 revealed eight patterns,
termed 1A to 1H (Fig. 3D). These patterns were themselves
species specific, or alternatively, specific variations
within a general DNA motif could be ascribed to one or more
species. Discrimination in the latter case required comparison
of partial sequence data from more than one Vc region (see
below).
Identification scheme for campylobacters based on partial
16S rDNA analysis. The distinct sequence patterns of the Vc
regions were used to develop a simplified scheme for the species-specific
identification of campylobacters by partial 16S
rDNA analysis. As shown in Table 3, most species displayed a
unique panel of DNA patterns, which enabled their unambiguous
identification. The exception was a lack of discrimination
among strains of C. jejuni and C. coli and atypical C. lari strains
(CF89-12, LMG 11760), which shared the pattern 6D-5D-2E-
1D. In addition, strains of C. hyointestinalis and C. lanienae,
which displayed the pattern 6D-5B/5C-2C-1B, could not be
discriminated.
DISCUSSION
The unambiguous identification of Campylobacter species is
difficult because these pathogens are slowly growing, fastidious
organisms which display only a few differential phenotypic
properties (36). Since automated DNA sequencing has become
generally available and the contents of public sequence databases
are constantly increasing, 16S rDNA analysis has become
a valuable tool for determination of the identities of bacterial
isolates (9, 18, 20, 24, 31). Therefore, we focused on 16S rDNA
sequencing to investigate its utility for the species-specific
identification of campylobacters.
Present guidelines suggest that 3% variation between two
rDNAs is the threshold at which two strains may be considered
to represent distinct species (7, 15, 24, 44). By taking this value
of sequence variation into account, the data derived from our
analysis of the whole-gene sequences is summarized as follows.
(i) Most Campylobacter species could clearly be differentiated,
since the minimum 16S rDNA sequence variation among the
most related taxa exceeded the 3% threshold (Table 2). (ii)
Lower levels of 16S rDNA variations were found between the
species C. rectus and C. showae (minimum diversity, 1.8%),
C. hyointestinalis and C. lanienae (minimum diversity, 1.9%),
C. helveticus and C. upsaliensis (minimum diversity, 1.6%),
C. hyointestinalis subsp. hyointestinalis and C. fetus (minimum
diversity, 1.6%), and classical (NARTC) C. lari strains and
C. jejuni-C. coli (minimum diversity, 1.6%). Nevertheless, in all
of these cases the interspecies variation significantly exceeded
the intraspecies variation (P 0.001) and the dendrogram
analysis revealed a species-specific clustering (Fig. 2). We conclude
that 16S rDNA-based differentiation of these species
displaying sequence diversities below 3% has practical application.
(iii) The limitation of the 16S rDNA analysis is the
inability to differentiate the species C. jejuni and C. coli and
atypical C. lari strains. Several C. jejuni and C. coli strains
shared identical 16S rDNA sequences, and nearly all strains of
these taxa were assigned to a common cluster (Fig. 2). In
addition, two atypical C. lari strains analyzed in this study were
also assigned to this cluster (Fig. 2). Their 16S rDNA sequences
displayed minimum diversities of 0.5% compared to
the sequences of C. coli and 0.6% compared to the sequences
of C. jejuni, whereas the maximum intraspecies diversity of C.
coli was 1.5% and that of C. jejuni was 0.4%. In contrast,
classical (NARTC) C. lari strains displayed higher degrees of
variation and could therefore be differentiated from this cluster
(Fig. 2). The observations that the members of the species
C. lari are phenotypically and genotypically diverse and that
the species may comprise multiple taxa are in concordance
with the findings presented in several other reports and highlight
the fact that the taxonomy of C. lari is still in progress (4,
10, 11, 12, 32, 37). Since C. jejuni, C. coli, and C. lari are
significant pathogens and their differentiation is important
when they are involved in clinical cases of infection, we suggest
the use of recently described PCR assays for accurate discrimination
and identification of the respective taxon (16, 29, 49,
50).
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