FIG. 3. (A) Alignment <strong>of</strong> campylobacter <strong>16S</strong> rDNA sequences within the Vc6 region revealed five distinct sequence patterns (patterns 6A to 6E). Only nucleotides different from those <strong>of</strong> C. fetus (pattern 6A) are indicated. (B) Alignment <strong>of</strong> campylobacter <strong>16S</strong> rDNA sequences within the Vc5 region revealed seven distinct sequence patterns (patterns 5A to 5G). Only nucleotides different from those <strong>of</strong> C. fetus (pattern 5A) are indicated. (C) Alignment <strong>of</strong> campylobacter <strong>16S</strong> rDNA sequences within the Vc2 region revealed 12 distinct sequence patterns (patterns 2A to 2L). Only nucleotides different from those <strong>of</strong> C. fetus (pattern 2A) are indicated. (D) Alignment <strong>of</strong> campylobacter <strong>16S</strong> rDNA sequences within the Vc1 region revealed eight distinct sequence patterns (patterns 1A to 1H). Only nucleotides different from those <strong>of</strong> C. fetus (pattern 1A) are indicated. Dashes indicate deletions at the respective base position. (A to D) a , nucleotide positions corresponding to the E. coli <strong>16S</strong> rRNA (5); b , nucleotides and positions <strong>of</strong> infrequently occurring polymorphisms within the sequence pattern. Downloaded from jcm.asm.org at UNIVERSITATSBIBLIOTHEK on June 1, 2010
VOL. 41, 2003 SPECIES-SPECIFIC IDENTIFICATION OF CAMPYLOBACTERS 2545 The identification <strong>of</strong> taxa to the subspecies level was possible for 14 <strong>of</strong> 15 strains <strong>of</strong> 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 <strong>of</strong> this strain remains unclear, we cannot recommend <strong>16S</strong> rDNA analysis as a singular method for the differentiation <strong>of</strong> C. hyointestinalis subspecies. A polyphasic approach that uses both phenotypic and genotypic methods should be used for identification <strong>of</strong> the subspecies (19). Subspecies-specific identification <strong>of</strong> the taxa C. jejuni and C. fetus <strong>by</strong> <strong>16S</strong> rDNA analysis was not possible (38). This study further shows that improved differentiation is possible <strong>by</strong> modification <strong>of</strong> <strong>16S</strong> rDNA analysis. For this purpose partial sequence data were used to determine species identities. The general structures <strong>of</strong> <strong>16S</strong> rRNAs and rDNAs comprise highly conserved and variable regions. Sequence alignments <strong>of</strong> the Campylobacter <strong>16S</strong> 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) <strong>of</strong> the procaryotic <strong>16S</strong> rRNA (rDNA) (35). The sequences <strong>of</strong> the Vc regions exhibited high levels <strong>of</strong> 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 <strong>of</strong> differentiation among the taxa C. coli and C. jejuni and atypical C. lari isolates, which had already been revealed <strong>by</strong> complete <strong>16S</strong> rDNA analysis. Analysis <strong>of</strong> 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 <strong>of</strong> the other gene sequences mentioned above. In addition, discrimination <strong>of</strong> certain isolates <strong>of</strong> 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 <strong>by</strong> complete <strong>16S</strong> rDNA sequence analysis. Moreover, it is significant that complete <strong>16S</strong> rDNA as well as analysis <strong>of</strong> 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 <strong>of</strong> several campylobacters, contributing to their misidentification as campylobacters <strong>by</strong> conventional phenotypic tests (1, 21, 36, 46, 52). We conclude that comparisons <strong>of</strong> <strong>16S</strong> rDNA sequences provide a substantially improved basis for the identification and differentiation <strong>of</strong> campylobacter species. Focused analysis <strong>of</strong> the variable regions <strong>of</strong>fers the ability to identify nearly the same range <strong>of</strong> species as whole-gene analysis, however, with the advantages <strong>of</strong> higher efficiency and lower cost. Although the significant pathogens C. jejuni, C. coli, and C. lari cannot be reliably discriminated <strong>by</strong> use <strong>of</strong> the <strong>16S</strong> rDNA data, the approach reported here <strong>of</strong>fers obvious advantages over existing methods. At present, no other singular method has the ability to identify such an extensive range <strong>of</strong> 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 <strong>of</strong> 47 campylobacter sequences to the database should prove valuable for clinical microbiologists using <strong>16S</strong> rDNA-based analysis during routine identification. In addition, we expect that the detailed description <strong>of</strong> the variable <strong>16S</strong> rDNA regions provided here will facilitate the design <strong>of</strong> 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. H<strong>of</strong>er and J. Flatscher (Mödling, Austria), and R. Krause, B. Ursinitsch, and K. Helleman (Graz, Austria). REFERENCES 1. al Rashid, S. T., I. Dakuna, H. Louie, D. Ng, P. Vandamme, W. Johnson, and V. L. Chan. 2000. <strong>Identification</strong> <strong>of</strong> Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, Arcobacter butzleri, and A. butzleri-like species based on the glyA gene. J. Clin. Microbiol. 38:1488–1494. 2. Bisping, W., and G. Amtsberg. 1988. Genus: Campylobacter, p.215–231. In Colour atlas for the diagnosis <strong>of</strong> bacterial pathogens in animals. Paul Parey Scientific Publishers, Berlin, Germany. 3. Blaser, M. J. 1997. Epidemiologic and clinical features <strong>of</strong> Campylobacter jejuni infections. J. Infect. Dis. 176:103–105. 4. Bolton, F. J., D. Coates, D. N. Hutchinson, and A. F. Godfree. 1987. A study <strong>of</strong> thermophilic campylobacters in a river system. J. Appl. Bacteriol. 62:167– 176. 5. Brosius, J., M. L. Palmer, P. J. Kennedy, and H. F. Noller. 1978. Complete nucleotide sequence <strong>of</strong> a <strong>16S</strong> ribosomal RNA gene from Escherichia coli. Proc. Natl. Acad. Sci. USA 75:4801–4805. 6. Cha, R. S., and W. G. Thilly. 1995. <strong>Specific</strong>ity, efficiency, and fidelity <strong>of</strong> PCR, p. 37–51. In C. W. Dieffenbach and G. S. Dveksler (ed.), PCR primer: a laboratory protocol. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 7. Clayton, R. A., G. Sutton, P. S. Hinkle, Jr., C. Bult, and C. Fields. 1995. Intraspecific variation in small-subunit rRNA sequences in GenBank: why single sequences may not adequately represent prokaryotic taxa. Int. J. Syst. Bacteriol. 45:595–599. 8. Denis, M., C. Soumet, K. Rivoal, G. Ermel, D. Blivet, G. Salvat, and P. Colin. 1999. Development <strong>of</strong> a m-PCR assay for simultaneous identification <strong>of</strong> Campylobacter jejuni and C. coli. Lett. Appl. Microbiol. 29:406–410. 9. Drancourt, M., C. Bollet, A. Carlioz, R. Martelin, J.-P. Gayral, and D. Raoult. 2000. <strong>16S</strong> ribosomal DNA sequence analysis <strong>of</strong> a large collection <strong>of</strong> environmental and clinical unidentifiable bacterial isolates. J. Clin. Microbiol. 38:3623–3630. 10. Duim, B., P. A. Vandamme, A. Rigter, S. Laevens, J. R. Dijkstra, and J. A. Wagenaar. 2001. Differentiation <strong>of</strong> Campylobacter species <strong>by</strong> AFLP fingerprinting. Microbiology 147:2729–2737. 11. Duim, B., J. A. Wagenaar, H. P. Endtz, J. R. Dijkstra, and P. A. R. Vandamme. 2001. <strong>Identification</strong> <strong>of</strong> distinct genogroups <strong>of</strong> C. lari with AFLP and protein pr<strong>of</strong>ile analysis. Int. J. Med. Microbiol. 291(Suppl. 31):143. 12. Endtz, H. P., J. S. Vliegenthart, P. Vandamme, H. W. Weverink, N. P. van den Braak, H. A. Verbrugh, and A. van Belkum. 1997. Genotypic diversity <strong>of</strong> Campylobacter lari isolated from mussels and oysters in The Netherlands. Int. J. Food Microbiol. 34:79–88. 13. Engberg, J., S. L. W. On, C. S. Harrington, and P. Gerner-Schmidt. 2000. Prevalence <strong>of</strong> Campylobacter, Arcobacter, Helicobacter, and Suturella spp. in human fecal samples as estimated <strong>by</strong> reevaluation <strong>of</strong> isolation methods for campylobacters. J. Clin. Microbiol. 38:286–291. 14. Etoh, Y., A. Yamamoto, and N. Goto. 1997. Intervening sequences in the <strong>16S</strong> rRNA genes <strong>of</strong> Campylobacter sp.: diversity <strong>of</strong> nucleotide sequences and uniformity <strong>of</strong> location. Microbiol. Immunol. 42:241–243. 15. Fox, G. E., J. D. Wisotzkey, and P. Jurtshuk, Jr. 1992. How close is close: <strong>16S</strong> rRNA sequence identity may not be sufficient to guarantee species identity. Int. J. Syst. Bacteriol. 42:166–170. 16. Gonzalez, I., K. A. Grant, P. T. Richardson, S. F. Park, and M. D. Collins. 1997. <strong>Specific</strong> identification <strong>of</strong> the enteropathogens Campylobacter jejuni and Campylobacter coli <strong>by</strong> using a PCR test based on the ceuE gene encoding a putative virulence determinant. J. Clin. Microbiol. 35:759–763. Downloaded from jcm.asm.org at UNIVERSITATSBIBLIOTHEK on June 1, 2010
Change language