2542 GORKIEWICZ ET AL. J. CLIN. MICROBIOL. Downloaded from jcm.asm.org at UNIVERSITATSBIBLIOTHEK on June 1, 2010 FIG. 2. Dendrogram <strong>of</strong> Campylobacter strains calculated from data for the nearly complete (94%) <strong>16S</strong> rDNA sequence. Analysis placed most sequences into species-specific clusters (shaded boxes). Strains which deviated from the species-specific clustering are indicated <strong>by</strong> asterisks. The scale bar at the top indicates a 1% difference in nucleotide sequence.
VOL. 41, 2003 SPECIES-SPECIFIC IDENTIFICATION OF CAMPYLOBACTERS 2543 TABLE 3. Pattern distribution among Campylobacter species <strong>Species</strong> 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 <strong>of</strong> C. coli (0.5%) and C. jejuni (0.6%) were significantly different from those <strong>of</strong> the classical C. lari strains compared to the sequences <strong>of</strong> C. coli and C. jejuni (1.6%) (P 0.001). The <strong>16S</strong> rDNA sequence diversities among Campylobacter species are given in Table 2. Characterization <strong>of</strong> variable regions within the <strong>16S</strong> rDNA. To improve the analysis, we investigated whether particular regions <strong>of</strong> <strong>16S</strong> rDNA yield sufficient information to discriminate among the taxa. <strong>16S</strong> rDNA alignment studies revealed four variable gene regions, which were termed Vc6, Vc5, Vc2, and Vc1, in accordance with the variable regions <strong>of</strong> the procaryotic <strong>16S</strong> rRNA. These regions displayed a high level <strong>of</strong> interspecies sequence variation. Among these we discerned several sequence patterns that are applicable for species-specific identification. Figures 3A to D show the alignments <strong>of</strong> the Campylobacter <strong>16S</strong> 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 <strong>of</strong> 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 <strong>of</strong> partial sequence data from more than one Vc region (see below). <strong>Identification</strong> scheme for campylobacters based on partial <strong>16S</strong> rDNA analysis. The distinct sequence patterns <strong>of</strong> the Vc regions were used to develop a simplified scheme for the species-specific identification <strong>of</strong> campylobacters <strong>by</strong> partial <strong>16S</strong> rDNA analysis. As shown in Table 3, most species displayed a unique panel <strong>of</strong> DNA patterns, which enabled their unambiguous identification. The exception was a lack <strong>of</strong> discrimination among strains <strong>of</strong> 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 <strong>of</strong> C. hyointestinalis and C. lanienae, which displayed the pattern 6D-5B/5C-2C-1B, could not be discriminated. DISCUSSION The unambiguous identification <strong>of</strong> 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 <strong>of</strong> public sequence databases are constantly increasing, <strong>16S</strong> rDNA analysis has become a valuable tool for determination <strong>of</strong> the identities <strong>of</strong> bacterial isolates (9, 18, 20, 24, 31). Therefore, we focused on <strong>16S</strong> rDNA sequencing to investigate its utility for the species-specific identification <strong>of</strong> 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 <strong>of</strong> sequence variation into account, the data derived from our analysis <strong>of</strong> the whole-gene sequences is summarized as follows. (i) Most Campylobacter species could clearly be differentiated, since the minimum <strong>16S</strong> rDNA sequence variation among the most related taxa exceeded the 3% threshold (Table 2). (ii) Lower levels <strong>of</strong> <strong>16S</strong> 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 <strong>of</strong> 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 <strong>16S</strong> rDNA-based differentiation <strong>of</strong> these species displaying sequence diversities below 3% has practical application. (iii) The limitation <strong>of</strong> the <strong>16S</strong> 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 <strong>16S</strong> rDNA sequences, and nearly all strains <strong>of</strong> 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 <strong>16S</strong> rDNA sequences displayed minimum diversities <strong>of</strong> 0.5% compared to the sequences <strong>of</strong> C. coli and 0.6% compared to the sequences <strong>of</strong> C. jejuni, whereas the maximum intraspecies diversity <strong>of</strong> C. coli was 1.5% and that <strong>of</strong> C. jejuni was 0.4%. In contrast, classical (NARTC) C. lari strains displayed higher degrees <strong>of</strong> variation and could therefore be differentiated from this cluster (Fig. 2). The observations that the members <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> infection, we suggest the use <strong>of</strong> recently described PCR assays for accurate discrimination and identification <strong>of</strong> the respective taxon (16, 29, 49, 50). Downloaded from jcm.asm.org at UNIVERSITATSBIBLIOTHEK on June 1, 2010
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