Resource Development and Investigation of Novel Species from Unidentified Pathogens in NCCP using MALDI-TOF MS and 16S rRNA Gene Analysis

Species identification is an important item to characterize unidentified bacterial pathogens in developing and managing bacterial resources. In this study, unidentified pathogens based on the results of an automated identification system were identified using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALD-TOF MS) and 16S rRNA gene analysis for development of national resources in the National Culture Collection for Pathogens (NCCP) in Korea. A total of 437 unidentified strains from branch banks of the NCCP were collected, and 16S rRNA and dnaJ gene sequencing, as well as MALDI-TOF MS analysis were performed to identify bacterial species. The mass spectra extracted were analyzed. Twelve strains exhibiting less than 98.65% similarity in 16S rRNA gene were selected as the primary candidates for novel species, and 21 strains exhibiting 98.65~99.0% similarity in 16S rRNA gene were selected as possible candidates for novel species. Among them, strain 32, belonging to Dermabacter sp., was finally selected as a possible strain representing a novel species and 14 unidentified bacterial strains using automated phenotypic identification system were newly registered at NCCP. The present study showed that unidentified pathogens using the automated phenotypic identification system were efficiently identified using the combination of MALDI-TOF MS and 16S rRNA gene analysis, and developed to the national resources in NCCP.

Taxonomic Identification of Bacillus Species Using Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry / 대한임상미생물학회지

BACKGROUND: In this study, we compared various methods of taxonomic identification of Bacillus strains: biochemical methods, 16S rRNA gene sequencing, and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). We also developed a pathogen- isolate resource database, thus increasing the identification rate when using MALDI-TOF MS. METHODS: Thirty Bacillus strains were obtained from the NCCP (National Culture Collection for Pathogens) and were identified using the VITEK 2 system (bio-Mérieux, France), API kit (bioMérieux, France), 16S rRNA gene sequencing, and MALDI-TOF MS. The pathogenicity of Bacillus cereus was confirmed through the identification of virulent genes using a multiplex PCR, and both protein extraction for protein profiling in MALDI-TOF MS and repetitive-sequence fingerprinting were performed. RESULTS: The identification rates at the species level were 40%, 80%, and 76.3% for the VITEK 2 system (bioMérieux), 16S rRNA gene sequencing, and MALDI-TOF MS, respectively. When the major spectrum-profiling dendrogram was compared with the phylogenetic tree, which was constructed based on the 16S rRNA gene sequences and rep-PCR fingerprinting, the classifications were confirmed to be effective. CONCLUSION: Identification of Bacillus strains using MALDI-TOF MS was more effective than that using the VITEK 2 system (bioMérieux), but was similar to that using 16S rRNA gene sequencing. Continual addition to a proteome-based database can result in increased identification rates for MALDI-TOF MS.

Molecular and Epidemiological Characterization of Carbapenem-Resistant Acinetobacter baumannii in Non-Tertiary Korean Hospitals

PURPOSE: The increasing prevalence and global spread of carbapenem-resistant Acinetobacter baumannii (A. baumannii) has become a serious problem. The aim of this study was to investigate molecular and epidemiological characteristics of carbapenem-resistant A. baumannii isolates collected from Korean non-tertiary hospitals. MATERIALS AND METHODS: Thirty six non-duplicated carbapenem-resistant A. baumannii isolates were collected from 17 non-tertiary hospitals in Korea between 2004 and 2006. Isolates were typed by multilocus sequence typing and repetitive-sequence-based PCR (rep-PCR). Detection of genes encoding OXA carbapenemase and their relationship with ISAba1 was performed by PCR. RESULTS: Two clones were prevalent among 36 isolates: ST69 (17 isolates, 47.2%) and ST92 (19 isolates, 52.8%). Rep-PCR patterns were diverse and revealed that all isolates were clustered into eight band patterns. The ISAba1-activated blaOXA-23-like and ISAba1-activated blaOXA-51-like genes were prevalent among the carbapenem-resistant A. baumannii isolates. CONCLUSION: The class D beta-lactamase genes of A. baumannii were distributed nationwide in non-tertiary Korean hospitals.

Orphan nuclear receptor small heterodimer partner inhibits angiotensin II-stimulated PAI-1 expression in vascular smooth muscle cells

Angiotensin II is a major effector molecule in the development of cardiovascular disease. In vascular smooth muscle cells (VSMCs), angiotensin II promotes cellular proliferation and extracellular matrix accumulation through the upregulation of plasminogen activator inhibitor-1 (PAI-1) expression. Previously, we demonstrated that small heterodimer partner (SHP) represses PAI-1 expression in the liver through the inhibition of TGF-beta signaling pathways. Here, we investigated whether SHP inhibited angiotensin II-stimulated PAI-1 expression in VSMCs. Adenovirus-mediated overexpression of SHP (Ad-SHP) in VSMCs inhibited angiotensin II- and TGF-beta-stimulated PAI-1 expression. Ad-SHP also inhibited angiotensin II-, TGF-beta- and Smad3-stimulated PAI-1 promoter activity, and angiotensin II-stimulated AP-1 activity. The level of PAI-1 expression was significantly higher in VSMCs of SHP-/- mice than wild type mice. Moreover, loss of SHP increased PAI-1 mRNA expression after angiotensin II treatment. These results suggest that SHP inhibits PAI-1 expression in VSMCs through the suppression of TGF-beta/Smad3 and AP-1 activity. Thus, agents that target the induction of SHP expression in VSMCs might help prevent the development and progression of atherosclerosis.

Outbreaks of Imipenem Resistant Acinetobacter Baumannii Producing OXA-23 beta-Lactamase in a Tertiary Care Hospital in Korea

PURPOSE: Since November 2006, imipenem-resistant Acinetobacter baumannii isolates have increased in Kyung Hee University Hospital in Seoul, Korea. The purpose of this study was to determine the genetic basis and molecular epidemiology of outbreak isolates. MATERIALS AND METHODS: Forty-nine non-repetitive isolates of the 734 IRAB strains were investigated in order to determine their characteristics. The modified Hodge and the ethylenediaminetetraacetic acid (EDTA)-disk synergy test were performed for the screening of carbapenemase and metallo-beta-lactamase production. Multiplex polymerase chain reaction (PCR) assays were performed for the detection of genes encoding for OXA-23-like, OXA-24-like, OXA-58-like and OXA-51-like carbapenemase. Pulsed-field gel electrophoresis (PFGE) was performed for strain identification. RESULTS: All isolates showed 100% resistance to ciprofloxacin and gentamicin, 97.9% resistance to cefepime, piperacillin/tazobactam, aztreonam, ceftazidime and piperacillin, 93.9% resistance to tobramycin and 57.1% resistance to amikacin. All of the 49 isolates (100%) showed positive results in the modified Hodge test and negative results in the EDTA-disk synergy test. They all (100%) possessed the encoding gene for an intrinsic OXA-51-like carbapenemase and an acquired OXA-23-like carbapenemase in the multiplex PCR assay. PFGE patterns revealed that all isolates were clonally related from A1 to A14. CONCLUSION: It is concluded that all of the 49 IRAB isolates acquired resistance to imipenem by producing OXA-23 carbapenemase and they might have originated from a common source.

Distribution of Genes Encoding Aminoglycoside Modifying Enzymes and Type Staphylococcal Chromosomal Cassette mec in Methicillin-resistant Staphylococcus aureus from Non-tertiary Hospitals

BACKGROUND: Many genes encoding aminoglycoside modifying enzymes (AMEs) on transposon or plasmid were transferred from one strain to another strain and inserted into a staphylococcal chromosomal cassette mec (SCCmec). There are very diverse subtypes in SCCmec type to the insertion of resistant genes. Therefore, we researched the resistance rates of antibiotics and distribution of AME genes according to SCCmec type in MRSA strains. MATERIALS AND METHODS: We isolated 640 Staphylococcus aureus from non-tertiary hospitals in 2004, detected mecA, aac(6')-aph(2"), aph(3')-IIIa, and ant(4')-Ia using the multiplex PCR method, tested antibacterial susceptibility disk diffusion and minimal inhibitory concentration, and determined SCCmec type. RESULTS: Of 640 S. aureus isolates, MRSA rate was 39.7% and all MRSA isolates carried mecA gene. Among 214 MRSA selected, aminoglycoside-resistant rates were 98.1% in kanamycin and tobramycin, 68.7% in gentamicin, 30.8% in amikacin, and 2.8% in netilmicin. The detection rates for aac(6')-aph(2"), aph(3')-IIIa, and ant(4')-Ia were 77.1%, 13.1%, and 53.3%, respectively. Also, SCCmec type was 50.9% in SCCmec type II, 16.4% in type III, and 32.7% in type IV. The genes encoding AMEs were distributed aac(6')-aph(2") (49.5%) and aac(6')-aph(2")/ant(4')-Ia (36.7%) in SCCmec type II, aph(3')-IIIa/aac(6')-aph(2") (60%) and aac(6')-aph(2") (31.4%) in type III, and aac(6')-aph(2")/ant(4')-Ia (41.4%) and ant(4')-Ia (50%) in type IV. CONCLUSION: 39.7% of S. aureus isolated from non-tertiary hospitals was resistant to methicillin. More than 90% of MRSA isolates were detected aac(6')-aph(2") in SCCmec type II and III, and ant(4')-Ia in type IV. With these results, the genes encoding AMEs may be closed related to SCCmec type.

Distribution of Genes Encoding Aminoglycoside Modifying Enzymes and Type Staphylococcal Chromosomal Cassette mec in Methicillin-resistant Staphylococcus aureus from Non-tertiary Hospitals

BACKGROUND: Many genes encoding aminoglycoside modifying enzymes (AMEs) on transposon or plasmid were transferred from one strain to another strain and inserted into a staphylococcal chromosomal cassette mec (SCCmec). There are very diverse subtypes in SCCmec type to the insertion of resistant genes. Therefore, we researched the resistance rates of antibiotics and distribution of AME genes according to SCCmec type in MRSA strains. MATERIALS AND METHODS: We isolated 640 Staphylococcus aureus from non-tertiary hospitals in 2004, detected mecA, aac(6')-aph(2"), aph(3')-IIIa, and ant(4')-Ia using the multiplex PCR method, tested antibacterial susceptibility disk diffusion and minimal inhibitory concentration, and determined SCCmec type. RESULTS: Of 640 S. aureus isolates, MRSA rate was 39.7% and all MRSA isolates carried mecA gene. Among 214 MRSA selected, aminoglycoside-resistant rates were 98.1% in kanamycin and tobramycin, 68.7% in gentamicin, 30.8% in amikacin, and 2.8% in netilmicin. The detection rates for aac(6')-aph(2"), aph(3')-IIIa, and ant(4')-Ia were 77.1%, 13.1%, and 53.3%, respectively. Also, SCCmec type was 50.9% in SCCmec type II, 16.4% in type III, and 32.7% in type IV. The genes encoding AMEs were distributed aac(6')-aph(2") (49.5%) and aac(6')-aph(2")/ant(4')-Ia (36.7%) in SCCmec type II, aph(3')-IIIa/aac(6')-aph(2") (60%) and aac(6')-aph(2") (31.4%) in type III, and aac(6')-aph(2")/ant(4')-Ia (41.4%) and ant(4')-Ia (50%) in type IV. CONCLUSION: 39.7% of S. aureus isolated from non-tertiary hospitals was resistant to methicillin. More than 90% of MRSA isolates were detected aac(6')-aph(2") in SCCmec type II and III, and ant(4')-Ia in type IV. With these results, the genes encoding AMEs may be closed related to SCCmec type.

Altered Expression of DNA Topoisomerase IIalpha, Ki-67, p53 and p27 in Non-Hodgkin's Lymphoma

BACKGROUND: Topoisomerase II (TOPO II) is an enzyme that separates intertwined chromosomes during DNA synthesis by transiently breaking and joining DNA strands. The level of TOP II is one of the determinants of cellular sensitivity to anti-tumor drugs in non-Hodgkin's lymphoma patients. The alpha form of TOPO II has been recently used as a marker of cellular proliferation. High levels of TOPO IIalpha are expressed in aggressive and proliferative tumors. METHODS: This study was designed to evaluate the relationship between TOPO IIalpha expression and clinicopathological parameters including age, gender, the serum LDH level, the serum beta2-microglobulin level and stage, or expressions, of Ki-67, p53 and p27, in non-Hodgkin's lymphoma. We analyzed forty-one biopsied tissue specimens from patients with non-Hodgkin's lymphoma. RESULTS: The expression of TOPO IIalpha increased with the clinical stage and it was correlated with Ki-67 and p53 expressions. However, TOPO IIalpha expression did not have any significant correlation with age, gender, the serum LDH level, the serum 2-microglobulin level and the p27 expression. CONCLUSIONS: TOPO IIalpha expression is a useful marker of cellular proliferation and it may serve as a prognostic factor of a tumor's progression and aggressiveness in non-Hodgkin's lymphomas.

Clinicopathologic Significance of CD44s, CD44v5 and CD44v6 Expression in Non Small Cell Lung Carcinomas

BACKGROUND: CD44 is a polymorphic family of transmembrane glycoproteins generated by alternative splicing of messenger RNA and is involved in the mechanism of tumor invasion and metastasis. METHODS: The expression of selected CD44 molecules (CD44s, CD44v5, and CD44v6) was determined immunohistochemically in 84 cases of non small cell lung carcinomas (NSCLCs). The results were compared with PCNA index, microvessel density (MVD), and clinicopathological parameters including patient? survival. RESULTS: CD44s showed a positive reaction in 61.9% (52/84) of NSCLCs, CD44v5 in 73.8% (62/84), and CD44v6 in 39.3% (33/84). Squamous cell carcinomas (SCCs) displayed preferential expression of all CD44 molecules in comparison with adenocarcinomas (ACs) (p<0.001). As a whole, the expression of CD44 molecules was not correlated with clinical parameters (stage, TNM-T, and TNM-N), PCNA index, or MVD. For ACs only, however, CD44v5 expression was negatively correlated with PCNA index (p<0.05). Poor survival was correlated with CD44v5 expression in ACs and CD44v6 in SCCs (both, p<0.05). CONCLUSIONS: These findings suggest that CD44 molecule in NSCLC could be a distinctive phenotypic marker for SCC, and the possibility that CD44v5 and CD44v6 are in some way instrumental in conditioning the biologic behavior of NSCLC according to major histologic types.

Morphometric Analysis of Glomeruli in the Experimental Rat Models of Hyperglycemia and Hyperfiltration / 대한신장학회잡지

OBJECTIVE: Diabetic nephropathy and ablation nephropathy are characterized by sclerotic processes in the glomeruli. To elucidate the site, degree and time-honored changes of glomerular sclerosis, morphometric analysis was performed using the experimental animals models. METHODS: The animals used were male Sprague Dowley rats and separated into 4 groups as young normal control, old control, streptozotocin-injected group, and right nephrectomized group. Chronologically kidney specimens were obtained after each treatment and processed to evaluated histologic changes. To evaluated the glomerular area, interstitial fibrosis and glomerular tuft fibrosis, the kidney specimens were fixed in Buin's solution, paraffin-embedded and 2 micrometer sections were Sirius red stained. To study the mesangial area, mesangial matrix area, glomerular basement membrane, and tubu lar basement membrane, the specimens were fixed in 2.5% glutaraldehyde, epon-embedded, double-stained and examined under the transmission electron microscope. All the specimens were analyzed morphometrically using the Image Pro Plus software. The obtained morphometric data were statistically analyzed to evaluate the differences of fibrotic processes and degree between experimental groups. RESULTS: Diabetic group revealed statistically significant increase of glomerular area from 8th week after streptozotocin injection to 24th week of experimental date. The parenchymal fibrosis and glomerular tuft fibrosis was prominent from the 2nd week of injection and steadily increased until the end of experimental date. The thickness of glomerular basement membrane was significantly increased even at the first week of injection and the tubular basement membrane also increased in thickness at the 3rd week of experiment. Ablation nephropathy model made by right nephrectomy showed increased glo merular area at the 7th week of ablation and the degree were intensified after 16th week of experimental date. The amount of stainable collagen in the renal parenchyme and glomerular tuft increased in the second week kidney sample and steadily increased thereafter until the end of experimental date. The increase of thickness of GBM and TBM also started to appear at the second week of operation. The old control also revealed fibrosis but the degree was less than the diabetic and ablation groups. Both diabetic and ablation nephropathy groups exhibited extensive increase of glomerular area, stainable colla gen, thickness of GBM and TBM at the end of experimental date and the ablation group revealed more extensive evidences of fibrosis without statistical significance. Comparison between the experimental groups were meaningless because the duration of the experimental manipulation was not the same. CONCLUSION: Glomerular and renal interstitial sclerosis and thickening of GBM and TBM are not the specific lesions of the diabetic glomerulopathy and are the common histologic changes occur in the kidney of partial parenchymal loss of any etiology. And it is suggested by this study that the common hemodynamic change involving the diabetic nephropathy, ablation nephropathy and physiologic aging is one of the important pathogenetic factors of glomerular sclerosis.

Morphometric Analysis of Glomeruli in the Experimental Rat Models of Hyperglycemia and Hyperfiltration / 대한신장학회잡지

OBJECTIVE: Diabetic nephropathy and ablation nephropathy are characterized by sclerotic processes in the glomeruli. To elucidate the site, degree and time-honored changes of glomerular sclerosis, morphometric analysis was performed using the experimental animals models. METHODS: The animals used were male Sprague Dowley rats and separated into 4 groups as young normal control, old control, streptozotocin-injected group, and right nephrectomized group. Chronologically kidney specimens were obtained after each treatment and processed to evaluated histologic changes. To evaluated the glomerular area, interstitial fibrosis and glomerular tuft fibrosis, the kidney specimens were fixed in Buin's solution, paraffin-embedded and 2 micrometer sections were Sirius red stained. To study the mesangial area, mesangial matrix area, glomerular basement membrane, and tubu lar basement membrane, the specimens were fixed in 2.5% glutaraldehyde, epon-embedded, double-stained and examined under the transmission electron microscope. All the specimens were analyzed morphometrically using the Image Pro Plus software. The obtained morphometric data were statistically analyzed to evaluate the differences of fibrotic processes and degree between experimental groups. RESULTS: Diabetic group revealed statistically significant increase of glomerular area from 8th week after streptozotocin injection to 24th week of experimental date. The parenchymal fibrosis and glomerular tuft fibrosis was prominent from the 2nd week of injection and steadily increased until the end of experimental date. The thickness of glomerular basement membrane was significantly increased even at the first week of injection and the tubular basement membrane also increased in thickness at the 3rd week of experiment. Ablation nephropathy model made by right nephrectomy showed increased glo merular area at the 7th week of ablation and the degree were intensified after 16th week of experimental date. The amount of stainable collagen in the renal parenchyme and glomerular tuft increased in the second week kidney sample and steadily increased thereafter until the end of experimental date. The increase of thickness of GBM and TBM also started to appear at the second week of operation. The old control also revealed fibrosis but the degree was less than the diabetic and ablation groups. Both diabetic and ablation nephropathy groups exhibited extensive increase of glomerular area, stainable colla gen, thickness of GBM and TBM at the end of experimental date and the ablation group revealed more extensive evidences of fibrosis without statistical significance. Comparison between the experimental groups were meaningless because the duration of the experimental manipulation was not the same. CONCLUSION: Glomerular and renal interstitial sclerosis and thickening of GBM and TBM are not the specific lesions of the diabetic glomerulopathy and are the common histologic changes occur in the kidney of partial parenchymal loss of any etiology. And it is suggested by this study that the common hemodynamic change involving the diabetic nephropathy, ablation nephropathy and physiologic aging is one of the important pathogenetic factors of glomerular sclerosis.