| Objective API20E strip test system is deemed to be golden standard in identifying Enterobacteriaceae. While, there exists some limit in identifying Yersina by API20E strip test. Except for some unstable biochemical reactions, different Yersina species have the same biochemical profile in API20E test system, for example, Yersinia ferderiksenii has the same biochemical profile with Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the 16S rRNA gene has different copies in prokaryotic organism and there is heterogeneity in different copies. So if using only one copy 16S rRNA gene to identify bacterium, it is likely to cause mistakes. Given to this, we devote to discuss the application of 16S rRNA gene in identifying Yersinia species by analyzing multiple copies of 16S rRNA gene.Methods In this study, we choose 744 Yersina strains from our laboratory and 24 complete-genome-sequenced strains from NCBI. Each of isolated strains is randomly selected five 16S rRNA gene clones through the method of vector T cloning, while 16S rRNA gene is obtained by random number table for each of complete-genome-sequenced strains. Then phylogenetic tree of 768 strains is established by combing five 16S rRNA gene of each strain. Clustering analysis of Yersinia spp. is made in the following.Result In this study, each strain is randomly selected five 16S rRNA gene clones for 768 Yersinia strains, so all together collected are 3,840 sequences of 16SrDNA gene from 10 species, which are divided into 439 patterns.The sequence similarity of pairwise copies in each Yersinia species strain exceeds 99%for most strains. There are nine strains with similarity below 98.7%. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. At the same time, we also find that different Yersinia species have the identical 16S rRNA gene sequences and each Yersinia species has its dominant 16S rRNA gene patterns.We summarized the number of 16S rRNA gene patterns for each strain. In general,60% of the strains have two or three types,18% have four, and 17% have one. Only 5% of the strains have five totally different 16S rRNA gene types. A comparison of the gene type number between the different Yersinia species is made. The number is primarily one or two in Yersinia pseudotuberculosis and Yersinia kristensenii, and two to four in Yersinia ferderiksenii/intermedia. The strains of pathogenic Yersinia enterocolitica usually have one to three types, whereas the non-pathogenic strains have two to three types. The proportion of Strains having five identical copies is 23.5% in non-pathogenic Yersinia enterocolitica strains, while only 8.6% in pathogenic strains. Yersinia pestis and Yersinia pseudotuberculosis do not have sequences with different copies.Conclusion A.16S rRNA gene of Yersina spp. has multiple copies among which there exists heterogeneity. Whereas, the discrepancy is below the threshold of 16S rRNA gene for identifying bacterial species, so it makes no difference when using only 16S rRNA gene to identify species. But it is likely to make an error for individual Yersinia strains with great difference among different copies. B. Conventional identification methods are needed in identifing different Yersinia species with identical 16S rRNA gene. C. Through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved. |
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