Yangsoon Lee

Identification of Novel Reference Genes Using Multiplatform Expression Data and Their Validation for Quantitative Gene Expression Analysis

Normalization of mRNA levels using endogenous reference genes (ERGs) is critical for an accurate comparison of gene expression between different samples. Despite the popularity of traditional ERGs (tERGs) such as GAPDH and ACTB, their expression variability in different tissues or disease status has been reported. Here, we first selected candidate housekeeping genes (HKGs) using human gene expression data from different platforms including EST, SAGE, and microarray, and 13 novel ERGs (nERGs) (ARL8B, CTBP1, CUL1, DIMT1L, FBXW2, GPBP1, LUC7L2, OAZ1, PAPOLA, SPG21, TRIM27, UBQLN1, ZNF207) were further identified from these HKGs. The mean coefficient variation (CV) values of nERGs were significantly lower than those of tERGs and the expression level of most nERGs was relatively lower than high expressing tERGs in all dataset. The higher expression stability and lower expression levels of most nERGs were validated in 108 human samples including formalin-fixed paraffin-embedded (FFPE) tissues, frozen tissues and cell lines, through quantitative real-time RT-PCR (qRT-PCR). Furthermore, the optimal number of nERGs required for accurate normalization was as few as two, while four genes were required when using tERGs in FFPE tissues. Most nERGs identified in this study should be better reference genes than tERGs, based on their higher expression stability and fewer numbers needed for normalization when multiple ERGs are required.

Prevalence and diversity of carbapenemases among imipenem-nonsusceptible Acinetobacter isolates in Korea: emergence of a novel OXA-182

Increase in multidrug-resistant Acinetobacter poses a serious problem in Korea. In this study, 190 imipenem (IPM)-nonsusceptible (NS) Acinetobacter isolates from 12 Korean hospitals in 2007 were used to determine species, prevalence, and antimicrobial susceptibility of OXA carbapenemase- and metallo-β-lactamase (MBL)-producing isolates. bla(OXA)-₂₃-like and ISAba1-asssociated bla(OXA)-₅₁-like genes were detected in 80% and 12% of 178 IPM-NS Acinetobacter baumannii isolates, respectively. A novel bla(OXA)-₁₈₂ was detected in 12 IPM-NS A. baumannii isolates. Twelve out of 14 MBL-producing isolates were non-baumanniiAcinetobacter. A. baumannii isolates with OXA carbapenemase were more often resistant to aminoglycosides, ciprofloxacin, and tigecycline than non-baumannii Acinetobacter isolates with MBL. Identical pulsed- field gel electrophoresis patterns were observed in 89% of A. baumannii isolates with bla(OXA)-₂₃-like gene. In conclusion, extremely rapid increase of IPM-NS A. baumannii in previous Korean studies was mainly due to clonal spread of OXA-23-producing A. baumannii isolates. A novel OXA-182 emerged in Korea.

Carbapenem-non-susceptible Acinetobacter baumannii of sequence type 92 or its single-locus variants with a G428T substitution in zone 2 of the rpoB gene

OBJECTIVES to investigate the epidemiological traits of carbapenem-non-susceptible Acinetobacter baumannii (CNSAB) and the usefulness of phylogenetic grouping based on partial rpoB gene sequencing in defining the epidemiological traits of CNSAB. METHODS a total of 547 non-duplicate clinical isolates of Acinetobacter spp. were collected from 19 hospitals in Korea in 2008. Detection of genes encoding OXA carbapenemases and metallo-β-lactamases was performed by PCR. The epidemiological relationships of the isolates were investigated by multilocus sequence typing and repetitive-sequence-based PCR. The 450 bp sequence (zone 2) of the rpoB gene was amplified and sequenced. RESULTS molecular characterization of the 272 CNSAB isolates identified five sequence types (STs): ST92, ST75, ST137, ST138 and ST69. The first four of these STs were clustered into clonal complex (CC) 92, sharing alleles at six of seven housekeeping gene loci; ST69 shared alleles at five of seven loci. CNSAB of CC92 carried the bla(OXA-23) gene (n = 169), the bla(OXA-51)-like gene preceded by ISAba1 (n = 89) or both (n = 14). Notably, all CNSAB isolates carried a G428T substitution in zone 2 of the rpoB gene. CONCLUSIONS CNSAB isolates of CC92 with the G428T substitution in zone 2 of the rpoB gene are disseminated nationwide in Korea. A. baumannii with the single nucleotide substitution may be more likely to acquire carbapenem resistance than are other isolates.

A Novel Insertion Sequence, ISAba10, Inserted into ISAba1 Adjacent to the blaOXA-23 Gene and Disrupting the Outer Membrane Protein Gene carO in Acinetobacter baumannii

ABSTRACT We investigated an outbreak caused by carbapenem-resistant Acinetobacter baumannii carrying the bla OXA-23 gene. A novel insertion sequence (IS), named ISAba10, was found to be inserted into the ISAba1 element preceding the bla OXA-23 gene in a group of isolates showing higher carbapenem MICs. The presence of ISAba10 was associated with increased OXA-23 expression, likely by providing additional promoter sequences. ISAba10 was also inserted into the carO outer membrane protein gene in most of these isolates.

Emergence of Clostridium difficile Ribotype 027 in Korea

Background Clostridium difficile infection (CDI) has markedly risen and is associated with hypervirulent ribotype 027 outbreaks in North America and Europe since 2003. The aims of this study were to determine the prevalence of ribotype 027 among C. difficile isolates in Korea, to characterize the ribotype 027 isolates, and to determine the clinical severity of CDI in patients infected with these isolates. Methods A total of 1,251 isolates of C. difficile recovered from stool specimens of suspected CDI patients at two tertiary-care hospitals and one commercial laboratory between 2002 and 2009. Genes for toxin A (tcdA), toxin B (tcdB), and binary toxin (cdtA and cdtB) were detected by PCR. Mutation in the tcdC gene was detected by sequencing after PCR amplification. For molecular genotyping, we performed PCR-ribotyping, pulsed-field gel electrophoresis (PFGE), and multilocus variable-number tandem-repeat analysis (MLVA). Minimum inhibitory concentrations of moxifloxacin were determined using Etest strips (AB bioMérieux, Sweden). Results We identified 7 isolates as ribotype 027. These isolates had the same tcdC mutation as the epidemic strain, and 6 of them were resistant to moxifloxacin. The isolates were categorized into 3 different PFGE types and 7 different MLVA types. All the 7 cases had occurred sporadically. Conclusions C. difficile ribotype 027 is uncommon, but it has emerged in Korea. The spread of this ribotype should be closely monitored in order to avoid an outbreak of CDI in Korea.

The changes of PCR ribotype and antimicrobial resistance of Clostridium difficile in a tertiary care hospital over 10 years

The aims of this study were to investigate any change in PCR ribotypes and to determine the antimicrobial resistance of common PCR ribotypes over a 10-year period in a tertiary care hospital. We conducted PCR ribotyping, antimicrobial susceptibility testing and DNA gyrase sequencing to identify changes in 1407 Clostridium difficile non-duplicated isolates obtained between 2000 and 2009. A total of 74 different ribotypes were found. The most prevalent ribotype was ribotype 001 (26.1 %). The prevalence of ribotype 017 was 17 % and that of ribotype 014/020 was 9.6 %. Ribotyping showed that the prevalence of ribotype 001 decreased and the prevalence of ribotypes 017, 014/020 and 018 increased over the 10 years. Antimicrobial resistance rates in prevalent ribotypes were: clindamycin, 81 %; cefotetan, 19 %; moxifloxacin, 42 %; imipenem, 8 %; ciprofloxacin, 100 % and erythromycin, 80 %. Ribotype 018 showed greater antimicrobial resistance than other ribotypes. All ribotype 018 strains showing moxifloxacin resistance had a substitution of a gyrA coding amino acid (Thr82 to Ile). This study will help the understanding of PCR ribotype trends and antimicrobial resistance of C. difficile in Korea.

Spread of CTX-M–type extended-spectrum β-lactamases among bloodstream isolates of Escherichia coli and Klebsiella pneumoniae from a Korean hospital

This study was to determine the prevalence and characteristics of CTX-M-type extended-spectrum beta-lactamases (ESBLs) in nonduplicate Escherichia coli (n=760) and Klebsiella pneumoniae (n=379) bloodstream isolates collected during January 2005 to October 2007 at a university hospital (2000 beds) in Seoul, Korea. Antimicrobial susceptibilities were determined by disk diffusion and agar dilution methods. The double-disk synergy test detected ESBLs in 8.7% (66/760) of E. coli and 11.3% (43/379) of K. pneumoniae isolates. Polymerase chain reaction detected bla(CTX-M) in 60/66 (90.9%) E. coli and 9/43 (20.9%) K. pneumoniae isolates with the ESBL phenotype. CTX-M-14 was the most common type of CTX-M ESBLs in both E. coli (n=32) and K. pneumoniae (n=6). CTX-M-15 was the 2nd most common type of CTX-M ESBLs in E. coli (n=22), but it was not detected in K. pneumoniae. In addition, CTX-M-24 (n=2), CTX-M-65 (n=2), CTX-M-27 (n=1), and CTX-M-32 (n=1) were detected for the 1st time in Korea.

Comparison of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry assay with conventional methods for detection of IMP-6, VIM-2, NDM-1, SIM-1, KPC-1, OXA-23, and OXA-51 carbapenemase-producing Acinetobacter spp., Pseudomonas aeruginosa, and Klebsiella pneumoniae.

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry assay was able to detect carbapenemase producers, including SIM-1 or OXA-51, within 4 hours using 20 μL of 0.5 g/L ertapenem solution as a substrate. This assay is more rapid and accurate than the modified Hodge test and 3-dimensional extract bioassay. Hence, it can be used an alternative test to identify carbapenemase-mediated carbapenem resistance in Gram-negative bacteria.

Antimicrobial Susceptibility Patterns for Recent Clinical Isolates of Anaerobic Bacteria in South Korea

ABSTRACT We determined the antimicrobial susceptibilities of 255 clinical isolates of anaerobic bacteria collected in 2007 and 2008 at a tertiary-care hospital in South Korea. Piperacillin-tazobactam, cefoxitin, imipenem, and meropenem were highly active β-lactam agents against most of the isolates tested. The rates of resistance of Bacteroides fragilis group organisms and anaerobic Gram-positive cocci to moxifloxacin were 11 to 18% and 0 to 27%, respectively.

Clonality and Resistome Analysis of KPC-Producing Klebsiella pneumoniae Strain Isolated in Korea Using Whole Genome Sequencing

We analyzed the whole genome sequence and resistome of the outbreak Klebsiella pneumoniae strain MP14 and compared it with those of K. pneumoniae carbapenemase- (KPC-) producing isolates that showed high similarity in the NCBI genome database. A KPC-2-producing multidrug-resistant (MDR) K. pneumoniae clinical isolate was obtained from a patient admitted to a Korean hospital in 2011. The strain MP14 was resistant to all tested β-lactams including monobactam, amikacin, levofloxacin, and cotrimoxazole, but susceptible to tigecycline and colistin. Resistome analysis showed the presence of β-lactamase genes including bla KPC-2, bla SHV-11, bla TEM-169, and bla OXA-9. MP14 also possessed aac(6′-)Ib, aadA2, and aph(3′-)Ia as aminoglycoside resistance-encoding genes, mph(A) for macrolides, oqxA and oqxB for quinolone, catA1 for phenicol, sul1 for sulfonamide, and dfrA12 for trimethoprim. Both SNP tree and cgMLST analysis showed the close relatedness with the KPC producers (KPNIH strains) isolated from an outbreak in the USA and colistin-resistant strains isolated in Italy. The plasmid-scaffold genes in plasmids pKpQil, pKpQil-IT, pKPN3, or pKPN-IT were identified in MP14, KPNIH, and Italian strains. The KPC-2-producing MDR K. pneumoniae ST258 stain isolated in Korea was highly clonally related with MDR K. pneumoniae strains from the USA and Italy. Global spread of KPC-producing K. pneumoniae is a worrying phenomenon.