Microbial Genomics Research: Sequencing of Klebsiella pneumoniae cps Region and Comparative Analysis

• Main Authors: Shu-Jui Hsu, 許書睿
• Other Authors: Shih-Feng Tsai
• Format: Others
• Language: en_US
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/34922435878820704322

碩士 === 國立陽明大學 === 生物醫學資訊研究所 === 98 === Klebsiella pneumoniae is a pathogen found in human’s gastrointestinal tract. Infection of K. pneumoniae potential can cause pyogenic liver abscess and septicemia. Infection of K. pneumoniae maybe community-acquired or hospital-acquired. The significant characteristics of K. pneumoniae include the thick capsule surrounding the cell wall and the virulence factor associated with the capsular polysaccharides (CPS) gene cluster. CPS is water soluble and commonly acidic with enormous structural diversity. Previous studies used serological detection method to determine different substrains that infect many different tissue including blood, urinary tract, and respiratory tract. To date, our group has sequenced the capsular polysaccharides gene cluster of seven clinical strains, as representative of serotypes K5, K9, K14, K62, NK8, NK29, and NK245. The genomic sequences of twelve cps are available in GenBank, including NTUH-K2044 (K1 serotype), Chedid (K2 serotype), MGH78578 (K52 serotype), A1142 (K57 serotype), and A1517. Comparative sequence analysis was performed using the MGCAT viewer to screen all available capsular polysaccharides genes. All 5’ regions have been predicted to share highly similar coding sequences from galF gene to wzc gene. 3’ regions were defined by the interval between two major genes gnd and ugd. The central region of the cluster genomic sequence from different strains was highly divergent. The cps of additional eight Klebsiella pneumoniae clinical isolates was sequenced in this study for serotype K16, K21, K28, K38, K53, K54, K55, and K57. We have devised a strain to work on three fragments as above to discover new cps sequences. The sequence of 5’ region and 3’ region were analyzed using general sequencing approach, whereas the central region were amplified using long range Polymerase chain reaction, followed by next-generation high throughput multiplex sequencing approach. All the cps 5’ regions have conserved gene, while the central region show high diversity, 3’ region has conserved gnd gene and ugd gene. These genes are involved in Klebsiella pneumoniae CPS synthesis. We also found two kinds of glycotransgerase (GT) gene in the central region and four kinds of gene pattern between 3’gnd gene and ugd gene that involved in. Our study supports that molecular genotyping method and comparative genomic hybridization are more powerful than general serotyping. The procedure reported here for accuracy CPS is efficient for detecting different substrains in a high throughput manner.