Effect of Intragenomic Sequence Heterogeneity among Multiple 16S rRNA Genes on Species Identification of Elizabethkingia

Accurate identification of Elizabethkingia species mostly requires the use of molecular techniques, and 16S rRNA gene sequencing is generally considered the method of choice. In this study, we evaluated the effect of intraspecific diversity among the multiple copies of the 16S rRNA gene on the accuracy of species identification in the genus Elizabethkingia. Sequences of 16S rRNA genes obtained from the 32 complete whole-genome sequences of Elizabethkingia deposited in GenBank and from 218 clinical isolates collected from 5 hospitals in Taiwan were analyzed. Four or five copies of 16S rRNA were identified in the Elizabethkingia species with complete genome sequences. The dissimilarity among the copies of the16S rRNA gene was,1% in all Elizabethkingia strains. E. meningoseptica demonstrated a significantly higher rate of nucleotide variations in the 16S rRNA than did E. anophelis (P = 0.011). Nucleotide alterations occurred more frequently in regions V2 and V6 than in other hypervariable regions (P, 0.001). E. meningoseptica, E. anophelis, and E. argenteiflava strains were clustered distinctly in the phylogenetic tree inferred from 16S rRNA genes, and the intragenomic variation of gene sequences had no profound effect on the classification of taxa. However, E. miricola, E. bruuniana, E. ursingii, and E. occulta were grouped closely in the phylogenetic analysis, and the variation among the multiple copies of the 16S rRNA in one E. ursingii strain affected species classification. Other marker genes may be required to supplement the species classification of closely related taxa in the genus Elizabethkingia. IMPORTANCE Incorrect identification of bacterial species would influence the epidemiology and clinical analysis of patients infected with Elizabethkingia. The results of the present study suggest that 16S rRNA gene sequencing should not be considered the gold standard for the accurate identification of Elizabethkingia species.