Phylogenetic analysis and PCR-restriction fragment length polymorphism identification of Campylobacter species based on partial groEL gene sequences

Abstract

The phylogeny of 12 Campylobacter species and reference strains of Arcobacter butzleri and Helicobacter pylori was studied based on partial 593-bp groEL gene sequences. The topology of the phylogenetic neighbor-joining tree based on the groEL gene was similar to that of the tree based on the 16S rRNA gene. However, groEL was found to provide a better resolution for Campylobacter species, with lower interspecies sequence similarities (range, 65 to 94%) compared with those for the 16S rRNA gene (range, 90 to 99%) and high intraspecies sequence similarities (range, 95 to 100%; average, 99%). A new universal reverse primer that amplifies a 517-bp fragment of the groEL gene was developed and used for PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of 68 strains representing 11 Campylobacter species as well as reference strains of A. butzlerii and H. pylori. Digestion with the AluI enzyme discriminated all Campylobacter species included in the study but showed more intraspecies diversity than digestion with the ApoI enzyme. A hippurate-negative variant of Campylobacter jejuni with a high level of groEL sequence similarity to both C. jejuni (96%) and C. coli (94%) gave a unique AluI profile and an ApoI profile identical to those of other C. jejuni strains. In conclusion, groEL gene sequencing and PCR-RFLP analysis are recommended as valuable tools for the identification of Campylobacter species.

FIG. 1.

Neighbor-joining tree based on partial 541-bp groEL sequences. The tree was rooted with H. pylori. The number at each branch point represents the percentage of bootstrap support calculated from 1,000 trees. The scale bar represents the sequence divergence.

FIG. 2.

Neighbor-joining tree based on the deduced partial 180-amino-acid GroEL sequences. The tree was rooted with H. pylori. The scale bar represents the sequence divergence.

FIG. 3.

RFLP analysis of the partial 517-bp groEL gene amplicon digested with AluI and ApoI. The combined dendrogram was derived with BioNumerics software by using the unweighted pair group method with arithmetic averages and the Dice coefficient with 1% optimization and tolerance.