Construction of a Sequence Database of 16S-23S rDNA Spacer (ITS) of Nonfermentative Gram-Negative Bacilli and Identification of Acinetobacter calcoaceticus-Acinetobacter baumannii (Acb) Complex by ITS Sequence Analysis

• Main Authors: Yu-Fang Wei, 魏郁芳
• Other Authors: Tsung-Chain Chang
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/04009168121218044858

碩士 === 國立成功大學 === 醫學工程研究所碩博士班 === 91 === Members of the genus Acinetobacter and other nonfermentative gram-negative bacteria are clinically important pathogens. However, clinical laboratories still lack simple methods that allow accurate identification of these species. For example, species of the Acinetobacter calcoaceticus- Acinetobacter baumannii complex (Acb complex) that includes Acinetobacter calcoaceticus (genospecies 1), A. baumannii (genospecies 2), unnamed genospecies 3 and 13TU are difficult to be differentiated. In fact, a complete discrimination between these species is possible only by DNA-DNA hybridization that is a complex and time-consuming method and cannot be performed in most clinical laboratories. In this study, the feasibility of using the sequences of 16S-23S rDNA spacer region (ITS) for the identification of Acb complex was evaluated. The ITS data in GenBank were insufficient to identify clinically relevant nonfermenters. The ITS regions from 108 strains (42 species) were amplified by PCR and sequenced; the sequence data in combination with those available in GenBank were used to construct an ITS sequence database for the identification of Acb complex. The database was used to test 82 clinical isolates of Acb complex; these microorgranisms included 63 isolates of A. baumannii and 19 isolates of A. calcoaceticus, as verified by API 20 NE. It is interesting to find among the 63 isolates of A. baumannii, 2 isolates might be genospecies 3 and 14 isolates were ungroupable, as revealed by ITS sequence analysis. Therefore, it is possible that 25.4% of clinical isolates of A. baumannii may be misidentified. Furthermore, among the 19 clinical isolates of A. calcoaceticus, 16 isolates might be genospecies 3 and 3 isolates were ungroupable as evidenced by ITS sequence analysis, and it seems that the designation of A. calcoaceticus to clinical isolates is, under most conditions, not correct. The 17 ungroupable isolates could be divided into 5 genotypes. The delineation of these ungroupable isolates, with their ITS sequences close to genospecies 2 and 13TU, needs further investigation such as DNA-DNA hybridization experiment. The ITS sequence database of nonfermenters may be useful for developing a DNA chip for identification of these microorganisms.