Species-specific identification of campylobacters by partial 16S rRNA gene sequencing

Abstract

Species-specific identification of campylobacters is problematic, primarily due to the absence of suitable biochemical assays and the existence of atypical strains. 16S rRNA gene (16S rDNA)-based identification of bacteria offers a possible alternative when phenotypic tests fail. Therefore, we evaluated the reliability of 16S rDNA sequencing for the species-specific identification of campylobacters. Sequence analyses were performed by using almost 94% of the complete 16S rRNA genes of 135 phenotypically characterized Campylobacter strains, including all known taxa of this genus. It was shown that 16S rDNA analysis enables specific identification of most Campylobacter species. The exception was a lack of discrimination among the taxa Campylobacter jejuni and C. coli and atypical C. lari strains, which shared identical or nearly identical 16S rDNA sequences. Subsequently, it was investigated whether partial 16S rDNA sequences are sufficient to determine species identity. Sequence alignments led to the identification of four 16S rDNA regions with high degrees of interspecies variation but with highly conserved sequence patterns within the respective species. A simple protocol based on the analysis of these sequence patterns was developed, which enabled the unambiguous identification of the majority of Campylobacter species. We recommend 16S rDNA sequence analysis as an effective, rapid procedure for the specific identification of campylobacters.

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.

FIG. 2.

Dendrogram of Campylobacter strains calculated from data for the nearly complete (94%) 16S rDNA sequence. Analysis placed most sequences into species-specific clusters (shaded boxes). Strains which deviated from the species-specific clustering are indicated by asterisks. The scale bar at the top indicates a 1% difference in nucleotide sequence.

FIG. 3.

(A) Alignment of campylobacter 16S rDNA sequences within the Vc6 region revealed five distinct sequence patterns (patterns 6A to 6E). Only nucleotides different from those of C. fetus (pattern 6A) are indicated. (B) Alignment of campylobacter 16S rDNA sequences within the Vc5 region revealed seven distinct sequence patterns (patterns 5A to 5G). Only nucleotides different from those of C. fetus (pattern 5A) are indicated. (C) Alignment of campylobacter 16S rDNA sequences within the Vc2 region revealed 12 distinct sequence patterns (patterns 2A to 2L). Only nucleotides different from those of C. fetus (pattern 2A) are indicated. (D) Alignment of campylobacter 16S rDNA sequences within the Vc1 region revealed eight distinct sequence patterns (patterns 1A to 1H). Only nucleotides different from those of 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 16S rRNA (5); ^b, nucleotides and positions of infrequently occurring polymorphisms within the sequence pattern.

FIG. 3.

(A) Alignment of campylobacter 16S rDNA sequences within the Vc6 region revealed five distinct sequence patterns (patterns 6A to 6E). Only nucleotides different from those of C. fetus (pattern 6A) are indicated. (B) Alignment of campylobacter 16S rDNA sequences within the Vc5 region revealed seven distinct sequence patterns (patterns 5A to 5G). Only nucleotides different from those of C. fetus (pattern 5A) are indicated. (C) Alignment of campylobacter 16S rDNA sequences within the Vc2 region revealed 12 distinct sequence patterns (patterns 2A to 2L). Only nucleotides different from those of C. fetus (pattern 2A) are indicated. (D) Alignment of campylobacter 16S rDNA sequences within the Vc1 region revealed eight distinct sequence patterns (patterns 1A to 1H). Only nucleotides different from those of 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 16S rRNA (5); ^b, nucleotides and positions of infrequently occurring polymorphisms within the sequence pattern.

FIG. 3.

(A) Alignment of campylobacter 16S rDNA sequences within the Vc6 region revealed five distinct sequence patterns (patterns 6A to 6E). Only nucleotides different from those of C. fetus (pattern 6A) are indicated. (B) Alignment of campylobacter 16S rDNA sequences within the Vc5 region revealed seven distinct sequence patterns (patterns 5A to 5G). Only nucleotides different from those of C. fetus (pattern 5A) are indicated. (C) Alignment of campylobacter 16S rDNA sequences within the Vc2 region revealed 12 distinct sequence patterns (patterns 2A to 2L). Only nucleotides different from those of C. fetus (pattern 2A) are indicated. (D) Alignment of campylobacter 16S rDNA sequences within the Vc1 region revealed eight distinct sequence patterns (patterns 1A to 1H). Only nucleotides different from those of 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 16S rRNA (5); ^b, nucleotides and positions of infrequently occurring polymorphisms within the sequence pattern.

FIG. 3.

(A) Alignment of campylobacter 16S rDNA sequences within the Vc6 region revealed five distinct sequence patterns (patterns 6A to 6E). Only nucleotides different from those of C. fetus (pattern 6A) are indicated. (B) Alignment of campylobacter 16S rDNA sequences within the Vc5 region revealed seven distinct sequence patterns (patterns 5A to 5G). Only nucleotides different from those of C. fetus (pattern 5A) are indicated. (C) Alignment of campylobacter 16S rDNA sequences within the Vc2 region revealed 12 distinct sequence patterns (patterns 2A to 2L). Only nucleotides different from those of C. fetus (pattern 2A) are indicated. (D) Alignment of campylobacter 16S rDNA sequences within the Vc1 region revealed eight distinct sequence patterns (patterns 1A to 1H). Only nucleotides different from those of 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 16S rRNA (5); ^b, nucleotides and positions of infrequently occurring polymorphisms within the sequence pattern.