Characterization of some bacterial strains isolated from animal clinical materials and identified as Corynebacterium xerosis by molecular biological techniques

Abstract

Eighteen Corynebacterium xerosis strains isolated from different animal clinical specimens were subjected to phenotypic and molecular genetic studies. On the basis of the results of the biochemical characterization, the strains were tentatively identified as C. xerosis. Phylogenetic analysis based on comparative analysis of the sequences of 16S rRNA and rpoB genes revealed that the 18 strains were highly related to C. xerosis, C. amycolatum, C. freneyi, and C. hansenii. There was a good concordance between 16S rRNA and partial rpoB gene sequencing results, although partial rpoB gene sequencing allowed better differentiation of C. xerosis. Alternatively, C. xerosis was also differentiated from C. freneyi and C. amycolatum by restriction fragment length polymorphism analysis of the 16S-23S rRNA gene intergenic spacer region. Phenotypic characterization indicated that besides acid production from D-turanose and 5-ketogluconate, 90% of the strains were able to reduce nitrate. The absence of the fatty acids C(14:0), C(15:0), C(16:1)omega 7c, and C(17:1)omega 8c can also facilitate the differentiation of C. xerosis from closely related species. The results of the present investigation demonstrated that for reliable identification of C. xerosis strains from clinical samples, a combination of phenotypic and molecular-biology-based identification techniques is necessary.

FIG. 1.

Digestion of the PCR-amplified 16S-23S rRNA gene IGS of Corynebacterium species using the restriction endonuclease CfoI. Lanes 1 to 22: C. freneyi CCUG 45704^T, animal strain M3, C. amycolatum CCUG 35685^T, C. hansenii CCUG 53252^T, C. xerosis CCUG 56051^T, and animal strains St36404, St33874, St34960, St36130, St38671, St85640, St53041, St51377/1, St53244, St51902, St51463, St51377/2, St51377/3, St46963, St49327, St47126, and St49485, respectively. Lanes M, molecular weight markers (100-bp ladder).

FIG. 2.

Dendrogram based on rpoB gene sequence comparison obtained with the neighbor-joining algorithm showing the phylogenetic relationship between animal strains of C. xerosis and the close relatives C. freneyi, C. amycolatum, and C. hansenii. Bootstrap values (expressed as percentages of 1,000 replications) higher than 50% are given at the branching points. Filled circles indicate that the corresponding nodes (groupings) are also obtained on the parsimony tree. Open circles indicate that the corresponding nodes (groupings) are also obtained on the maximum-likelihood and parsimony trees. Corynebacterium bovis CIP 54.80^T (AY492236) was used as an outgroup. Bar, 1% sequence divergence.

FIG. 3.

Dendrogram based on 16S rRNA gene sequence comparison obtained with the neighbor-joining algorithm showing the phylogenetic relationship between animal strains of C. xerosis and the close relatives C. freneyi, C. amycolatum, and C. hansenii. Bootstrap values (expressed as percentages of 1,000 replications) higher than 50% are given at the branching points. Filled circles indicate that the corresponding nodes (groupings) are also obtained on the parsimony tree. Open circles indicate that the corresponding nodes (groupings) are also obtained on the maximum-likelihood and parsimony trees. C. bovis NCTC 3324^T (X82051) was used as an outgroup. Bar, 1% sequence divergence.