Development Of Whole-genome DNA Microarray Of Klebsiella Pneumoniae And Stuty Of Large Variably-presented Gene Cluster (LVPC)-based Genotyping

Klebsiella pneumoniae is a gram-negative. It is ubiquitous in thenatural environment, and is primary in the respiratory and intestinal tract ofhumans and animals. K. pneumoniae is an opportunistic pathogenassociated with both community-acquired and hospital-acquired infections,including septicemia, pneumonia, urinary tract infections, and meningitis. Itis also the second most common microorganism isolated, after Escherichiacoli, from patients with gram-negative bacteria. Over the past2decades, anew type of community-acquired K. pneumoniae associated with pyogenicliver abscess has been reported to occur in Taiwan, Japan, Europe, NorthAmerica, and Korea. This emerging disease is often complicated with septicmeningitis and endophthalmitis.In recent years, with the rapid development of molecular biologytechniques，greatly promote us research K. pneumoniae at the gene level. Inthis study, through the complete genome sequence of the K. pneumoniae, todevelop the whole-genome DNA microarray and establish a method of microarray-based comparative genomic analysis of K. pneumoniae.Through the genotyping of LVPC，to identify the differences betweendifferent strains of K. pneumoniae accordingly. Finally, comprehensiveanalysis of the K. pneumoniae genomic polymorphism，laid the foundationto investigate the population structure and genome microevolution of K.pneumoniae.Development of whole-genome DNA microarray of KlebsiellapneumoniaeBased on the genomic sequences of K. pneumoniae NTUH-2044,5075genes were chosen, which were amplified by PCR and printed onto glassslides. Three sets of two-fluorescence comparative hybridizations wereperformed to evaluate the quality of the microarray. Finally, thewhole-genome DNA microarray of K. pneumoniae was successfullydeveloped, and a method of microarray-based comparative genomicanalysis of K. pneumoniae was established accordingly.Large variably-presented gene cluster (LVPC)-based genotypingComparative genomics analysis was conducted for the four clinicalK. pneumoniae strains with the determined complete genome sequences,namely NTUH-K2044, MGH78578, HS11286, and KCTC2242. Coregenes were define as those conserved (identity>80%plus coverage>80%)among all the four genomes, and then a total of44candidates of largevariably-presented gene cluster (LVPC, a genomic region containing at least ten consecutive genes and variably distributed within the genomes ofdifferent strains) were identified from the pool of non-redundantaccessory genes. A single gene was chosen from each LVPC and thegene-specific primer pairs were designed to perform the PCR-basedscreening within the82strains tested. According to the binary LVPC data, atotal of30different LVPC profiles were identified，these82strains could beassigned into six complexes. Strains of the same serotype clusteringtogether, and the different serotypes distributed in different clusters,showing genetic polymorphism of different strains. LVPC-basedgenotyping was simple and inexpensive, rapidly genotyped a large numberof strains. However, the resolution of the method was limited, so you cancombine with another genotyping method.