Allele-specific duplex polymerase chain reaction to differentiate Mycobacterium abscessus subspecies and to detect highly clarithromycin-resistant isolates.

On the basis of the structural differences of erm, we used a duplex polymerase chain reaction (PCR) to differentiate Mycobacterium abscessus subsp. abscessus and subsp. massiliense isolates and to detect the point mutations of 23S rRNA gene that confer a high level of resistance to clarithromycin. Subsp. massiliense strains occupying almost half of the clinical isolates can be simply identified, and their clarithromycin susceptibility can be rapidly determined.

Involvement of miR-9/MCPIP1 axis in PDGF-BB-mediated neurogenesis in neuronal progenitor cells.

Highly conserved microRNA-9 (miR-9) has a critical role in various cellular processes including neurogenesis. However, its regulation by neurotropins that are known to mediate neurogenesis remains poorly defined. In this study, we identify platelet-derived growth factor-BB (PDGF-BB)-mediated upregulation of miR-9, which in turn downregulates its target gene monocyte chemotactic protein-induced protein 1 (MCPIP1), as a key player in modulating proliferation, neuronal differentiation as well as migration of neuronal progenitor cells (NPCs). Results indicate that miR-9-mediated NPC proliferation and neuronal differentiation involves signaling via the nuclear factor-kappa B (NF-κB) and cAMP response element-binding protein (CREB) pathways, and that NPC migration involves CREB but not the NF-κB signaling. These findings thus suggest that miR-9-mediated downregulation of MCPIP1 acts as a molecular switch regulation of neurogenesis.

Patterns of pncA mutations in drug-resistant Mycobacterium tuberculosis isolated from patients in South Korea.

BACKGROUND: Pyrazinamide (PZA), one of the most effective anti-tuberculosis drugs, becomes toxic to Mycobacterium tuberculosis when converted to pyrazinoic acid by pyrazinamidase (PZase). PZA resistance is caused mainly by the loss of enzyme activity by mutation. OBJECTIVE: To investigate the patterns of pncA mutations in PZA-resistant mycobacteria isolated from South Korean patients. METHODS: Mycobacterial isolates with clinically proven drug resistance were cultured to determine susceptibility to anti-tuberculosis agents. pncA mutations were recognised by sequencing and compared with the relevant wild-type DNA sequence. RESULTS: Among 108 isolates, 102 were successfully cultured and underwent drug susceptibility testing; all were multidrug-resistant (MDR). pncA mutations were found in 86 cultured isolates (85.1%): 55 (84.6%) in MDR and 31 (86.1%) in extensively drug-resistant isolates. Substitution of a single nucleotide was most common. The most frequent mutations were a deletion that caused a frameshift at nucleotide (nt) 71, a substitution at nt 403 and a substitution at nt 11. Combined, these accounted for ≈ 40% of all mutations. However, 15 samples (14.9%) with defective PZase activity showed no mutation. CONCLUSION: pncA mutation in M. tuberculosis is a major mechanism of PZA resistance in MDR isolates from patients in South Korea. The patterns of mutation might be more scattered and diverse. DNA-based diagnosis of PZA resistance has potential for the rapid detection of drug resistance.

Elevated levels of macrophage migration inhibitory factor in women with metabolic syndrome.

Metabolic syndrome is a complex clinical disorder characterized by obesity, a disturbance of glucose metabolism, dyslipidemia, and hypertension, leading to increased cardiovascular risk. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced both by innate immune cells and by adipocytes, and it plays an important role in inflammatory and cardiovascular diseases. The goal of this study was to evaluate the expression of circulating MIF in patients with metabolic syndrome. A study was conducted involving 172 persons who attended the Jeju National University Hospital Health Promotion Center. Among the 172 subjects, 88 patients with metabolic syndrome and 84 healthy control subjects were included. Serum MIF levels were considerably higher in patients with metabolic syndrome than in healthy subjects (mean±SEM, 1413.0-pg/ml±102.6 vs. 1077.0-pg/ml±-91.3, p=0.016). Among the metabolic syndrome patients, MIF levels were significantly increased in women (1403.0-pg/ml±114.2 vs. 921.3 pg/ml±117.3, p=0.005), but not in men. Even after further linear regression adjustment for age and body mass index, the expression of MIF for women with metabolic syndrome was still clearly elevated when compared to healthy subjects (p=0.011). Circulating MIF concentrations showed a gender disparity between healthy and metabolic syndrome subjects. An elevation of systemic MIF in women with metabolic syndrome may contribute to pathogenesis of metabolic syndrome or to the development of metabolic syndrome-related diseases, such as atherosclerosis and type 2 diabetes mellitus.

Comparative assays of the rpoB gene for identification of Mycobacterium tuberculosis isolated from patients in Sudan.

OBJECTIVES: To characterise mycobacterial clinical isolates based on amplification of the rpoB gene. SETTING: One hundred and thirty-five mycobacterial isolates cultured from suspected pulmonary tuberculosis (TB) patients were identified phenotypically. Molecular characterisation of the isolates was performed based on amplification of the rpoB gene, using duplex polymerase chain reaction (DPCR), PCR-restriction fragment length polymorphism (RFLP) and nested PCR-based sequence analysis techniques. RESULTS: The DPCR assay identified 129 of 135 (95.5%) clinical isolates as Mycobacterium tuberculosis complex species. Restriction enzyme analysis of the rpoB PCR product using Hind II identified 134 of the 135 (99.3%) isolates as M. tuberculosis complex, while nested PCR sequence analysis of the rpoB gene identified 133/133 examined isolates (100%) as M. tuberculosis species. No mycobacteria other than M. tuberculosis (MOTT) were detected among the studied isolates. CONCLUSION: DPCR, PCR/RFLP Hind II and nested PCR sequence analysis of the rpoB gene techniques showed comparable efficiency in the characterisation of Mycobacterium isolates. Nested PCR sequence analysis of the rpoB gene was superior to PCR/RFLP for characterisation of suspected M. tuberculosis isolates, while the DPCR technique showed less sensitivity. As PCR-RFLP requires less sophisticated laboratory facilities than nested PCR sequence analysis, it would be more appropriate to be adopted for accurate characterisation of mycobacteria in countries with a weak infrastructure.

RNA polymerase beta-subunit gene (rpoB) sequence analysis for the identification of Bacteroides spp.

Partial rpoB sequences (317 bp) of 11 species of Bacteroides, two Porphyromonas spp. and two Prevotella spp. were compared to delineate the genetic relationships among Bacteroides and closely related anaerobic species. The high level of inter-species sequence dissimilarities (7.6-20.8%) allowed the various Bacteroides spp. to be distinguished. The position of the Bacteroides distasonis and Bacteriodes merdae cluster in the rpoB tree was different from the position in the 16S rRNA gene tree. Based on rpoB sequence similarity and clustering in the rpoB tree, it was possible to correctly re-identify 80 clinical isolates of Bacteroides. In addition to two subgroups, cfiA-negative (division I) and cfiA-positive (division II), of Bacteroides fragilis isolates, two distinct subgroups were also found among Bacteroides ovatus and Bacteroides thetaiotaomicron isolates. Bacteroides genus-specific rpoB PCR and B. fragilis species-specific rpoB PCR allowed Bacteroides spp. to be differentiated from Porphyromonas and Prevotella spp., and also allowed B. fragilis to be differentiated from other non-fragilisBacteroides spp. included in the present study.

Genetic diversity of Legionella pneumophila inferred from rpoB and dotA sequences.

This study characterised the population structure of Legionella pneumophila by comparing the rpoB (300-bp) and dotA (360-bp) sequences of 267 isolates (18 reference strains, 149 Korean isolates and 100 Japanese isolates). In addition to the six clonal subgroups established previously, four subgroups, P-V to P-VIII, were identified. Subgroupings based on rpoB and dotA sequences were found to correlate with the source of the isolates, and this data may be useful for future epidemiological studies. Fourteen (five Korean and nine Japanese) isolates showed incongruent subgroupings in the rpoB and dotA trees, suggesting that genetic exchange among subgroups, and even among subspecies, may occur frequently in nature.

Cross-resistance between rifampicin and KRM-1648 is associated with specific rpoB alleles in Mycobacterium tuberculosis.

KRM-1648 resistant Mycobacterium tuberculosis strains were identified from a collection of rifampicin-resistant strains. Several strains had novel rpoB gene mutations in codons 512, 529 and 533 of the rpoB gene. The strains with mutations in codons 526 or 531, major mutation sites in rifampicin-resistant M. tuberculosis, were resistant to KRM-1648. Also, the strains with other mutations in the rpoB gene that were initially susceptible to KRM-1648 were prone to developing KRM-1648 resistance after further mutation. Thus, KRM-1648 is unlikely to be useful for the treatment of rifampicin-resistant tuberculosis.

Respiratory syncytial virus infection induces matrix metalloproteinase-9 expression in epithelial cells.

Increased gelatinolytic activity was observed in respiratory syncytial virus (RSV)-infected HEp-2 cells by using zymography. The anti-matrix metalloproteinase-9 (MMP-9) antibody specifically reduced the gelatinolytic activity suggesting that the increased gelatinolytic activity was due to the MMP-9. It was also supported by the results from immunofluorescent staining, treatment of MMP inhibitors, and RSV infection of the cell clones that were transfected with plasmids to express more MMP-9 and tissue type inhibitor of metalloproteinase-1 (TIMP-1). The gelatinolytic activity of extracellular MMP-9 in RSV-infected HEp-2 cells increased 1.5 +/- 0.2 fold compared with the control (p < 0.01). Cell surface MMP-9 expression was also clearly detected by immunofluorescent staining. Treatment with 1,10-phenanthroline (0.05 mM), ethylenediamine-tetraacetate (EDTA) (1.5 mM), and penta-O-galloyl-beta-D-glucose (PGG) (3.3 microM) inhibited RSV multiplication as well as syncytia formation. Furthermore, the average syncytia size increased when the cells expressing more MMP-9 were infected by RSV. In contrast, syncytia formation was inhibited in the cells manipulated to express TIMP-1. Thus, this study concludes that although RSV infection induces MMP-9, which can enhance the syncytia formation leading to RSV multiplication and spread it can be inhibited by MMP inhibitors.

Detection of rifampin-resistant Mycobacterium tuberculosis in sputa by nested PCR-linked single-strand conformation polymorphism and DNA sequencing.

Either PCR-mediated single strand conformation polymorphism (SSCP) analysis or DNA sequencing of rpoB DNA (157 bp) can be used as a rapid screening method for the detection of mutations related to the rifampin resistance of Mycobacterium tuberculosis. However, due to the nonspecific amplification of rpoB DNA from nontuberculous mycobacteria these methods cannot be directly applied to clinical specimens such as sputa. We developed a nested PCR method that can specifically amplify the rpoB DNA of M. tuberculosis on the basis of rpoB DNA sequences of 44 mycobacteria. Nested PCR-linked SSCP analysis and the DNA sequencing method were applied directly in order to detect M. tuberculosis and determine its rifampin susceptibility in 56 sputa. The results obtained by nested PCR-SSCP and DNA sequencing were concordant with those of conventional drug susceptibility testing and DNA sequencing performed with culture isolates.

Differentiation of mycobacterial species by PCR-restriction analysis of DNA (342 base pairs) of the RNA polymerase gene (rpoB).

PCR amplification-restriction analysis (PRA) of rpoB DNA (342 bp), which comprises the Rif(r) region, was used for the differential identification of 49 mycobacteria. The DNA had been used previously for the identification of mycobacterial species by comparative sequence analysis (B. J. Kim et al., J. Clin. Microbiol. 37:1714-1720, 1999). Digestion with four restriction enzymes (HaeIII, HindII, MvaI, and AccII), which were selected on the basis of rpoB DNA sequences, generated distinctive PRA patterns that allowed not only the reference strains but also the clinical isolates of mycobacteria to be distinguished. Both rapidly and slowly growing mycobacteria were distinctly differentiated by HaeIII digestion of the amplified rpoB DNA. By HindII digestion the Mycobacterium tuberculosis complex was distinguished from the other mycobacteria. Furthermore, six subspecies of Mycobacterium kansasii (subspecies I to VI) as well as the closely related Mycobacterium gastri, and other closely related species, were distinguished by simultaneous digestion of MvaI and AccII. According to the rpoB PRA scheme, 240 strains of clinical isolates could be identified. It was also possible to detect and identify M. tuberculosis directly from sputa and bronchoalveolar lavage specimens. These results suggest that PRA of rpoB DNA is a simple and feasible method not only for the differentiation of culture isolates but also for the rapid detection and identification of pathogenic mycobacteria in primary clinical specimens.

Multidrug-resistant Salmonella typhimurium and Salmonella enteritidis identified by multiplex PCR from animals.

Antibiotic resistance in Salmonella enteritidis and S. typhimurium, one of most frequent etiologic pathogens of food-borne bacterial gastroenteritidis in humans, is a serious health problem worldwide. Fifteen and 22 each of S. enteritidis and S. typhimurium were isolated from animals from 1983 to 1999 in Korea and tested for their antibiotic resistance patterns and phage types. S. enteritides isolates were highly resistant to sulfonamides (86.7%) and four of them (26.6%) showed multiple antibiotic resistance. The most frequent phage type (PT) of S. enteritids was PT1 (33.3%) even though none of them had multiple antibiotic resistance. S. typhimurium isolates were highly resistant to streptomycin, sulfonamides, and tetracycline, 100%, 95.5%, and 86.4% respectively. The incidence of multiple antibiotic resistance of S. typhimurium isolates was extremely high (100%) comparing to S. enteritidis isolates (26.7%). Two of the five ACSSuT type S. typhimurium isolates, resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline, were phage type DT104. All S. typhimurium isolates were sensitive to florfenicol. For the rapid detection of multiple antibiotic resistant S. enteritidis and S. typhimurium isolates, particularly ACSSuT type S. typhimurium DT104, antibiotic resistance genes, cmlA/tetR, PSE-1, and TEM, and Salmonella spp. Specific gene, SipB/C, were amplified using four pairs of primers in hot-started multiplex polymerase chain reaction. Two Korean isolates of S. typhimurium DT104 showed TEM amplicons instead of PSE-1 for the ampicillin resistance. The multiplex PCR used in this study was useful in rapid detection of ACSSuT type S. typhimurium and identification of b-lactamase gene distribution among Salmonella isolates.

Differentiation of Borrelia burgdorferi sensu lato on the basis of RNA polymerase gene (rpoB) sequences.

We determined the nucleotide sequences (329 bp) of the rpoB DNAs from 22 reference strains of Borrelia. No insertions or deletions were observed. Deduced amino acid sequences of amplified rpoB DNA comprised 109 amino acid residues (N(450) to M(558) [Escherichia coli numbering]). All amino acid sequences were identical with the exception of those of Borrelia lusitaniae PotiB2 (T(461)-->A) and B. bissettii DN127 (I(498)-->V). Each species of B. burgdorferi sensu lato was differentiated as a distinct entity in the phylogenetic tree constructed by a neighbor-joining method. B. burgdorferi sensu lato could be distinguished from B. turicatae and B. hermsii, which are associated with relapsing fever. Seventeen Korean isolates could be identified by PCR-linked direct sequencing and restriction analysis of the rpoB DNA. These results suggest that rpoB DNA is useful for identification and characterization of Borrelia. In addition, we developed the rapid species identification method using the species-specific primer sets based on rpoB gene sequences.

Sporotrichoid dermatosis caused by Mycobacterium abscessus from a public bath.

Infections caused by nontuberculous mycobacteria (NTM) are usually associated with immunocompromised states. More recently, however, NTM infections are being diagnosed with greater frequency in patients lacking traditional risk factors. However, cutaneous infection with rapidly growing mycobacteria is uncommon, and diagnosis may be difficult. Herein we present a case of sporotrichoid dermatosis on both forearms caused by Mycobacterium abscessus in a 34-year-old female (case 1). Mycobacterium abscesus was identified by culture as a colorless colony with rapid growth and by comparative sequence analysis of the rpoB gene. The patient was suspected to have been infected in a public bath in which she worked, it was located in a famous hot spring area in Korea. The condition was first noticed after she had been working in the bath for two years and after another employee (case 2) suffered similar lesions which had responded to treatment. The patient's skin lesions were successfully treated with anti-tuberculous drugs for six months.

Characterization of Borrelia burgdorferi strains isolated from Korea by 16S rDNA sequence analysis and PCR-RFLP analysis of rrf (5S)-rrl (23S) intergenic spacer amplicons.

Haenam strains of Borrelia burgdorferi, which had been isolated from Ixodes granulatus and Apodemus agrarius in Haenam, Korea, were characterized by PCR-RFLP analysis of rrf (5S)-rrl (23S) intergenic spacer amplicons and by sequence analysis of the 16S rRNA gene (rDNA). The Msel and Dral restriction patterns of the 5S-23S intergenic spacer amplicons of Haenam strains differed from those of other B. burgdorferi sensu lato strains. Furthermore, in the phylogenetic tree based on the 16S rDNA sequences, Haenam strains formed a distinctive cluster, clearly separated from the other members of B. burgdorferi sensu lato. These results suggest that, apart from Borrelia garinii and Borrelia afzelii, other genotypes of B. burgdorferi sensu lato exist in Korea and the Haenam strain is a newly identified one.

Sp1 mediates constitutive and transforming growth factor beta-inducible expression of urokinase type plasminogen activator receptor gene in human monocyte-like U937 cells.

Urokinase type plasminogen activator receptor (uPAR) is known to be involved in conversion of plasminogen into plasmin and its expression can be regulated by a variety of biological agents including transforming growth factor beta (TGF-beta). In the present study, we cloned the promoter region of the human uPAR (huPAR) gene (-653 to +61) and investigated the transcription regulatory mechanism of the expression of the huPAR gene upon treatment with TGF-beta in human monocyte-like U937 cells. By deletion and point mutational analysis of the huPAR gene promoter, it was found that the sequence positioned at -70 is required for both constitutive and TGF-beta-inducible expression of the huPAR gene in U937 cells. Using electrophoretic mobility shift assay, we could observe that Sp1 formed a DNA-protein complex at the -70 sequence. In addition, antisense oligonucleotide against human Sp1 blocked both constitutive and TGF-beta-inducible expression of the luciferase reporter gene driven by the huPAR gene promoter in U937 cells. These results led us to conclude that Sp1 transcription factor mediates constitutive and TGF-beta-inducible expression of the huPAR gene in U937 cells through binding to the sequence located at -70.

Antiviral effects of 28-deacetylsendanin on herpes simplex virus-1 replication.

The compound purified from the fruit of Melia azedarach exerted an antiviral effect on herpes simplex virus-1 (HSV-1) in Vero cells. It was identified as 28-deacetylsendanin (28-DAS). The 50% inhibitory concentration (IC50) of 28-DAS was 1.46 microg/ml without cytotoxicity at 400 microg/ml on Vero cells. Electron microscopy showed that low electron-dense cores of newly synthesized nucleocapsids remained in swollen nuclei and no extracellular virus particles were observed at 15 h p.i. Consistent with this result, it was confirmed by a plaque assay that few infectious progeny viruses were released from the 28-DAS-treated virus-infected cells at 24 h p.i. Intracellular viruses in 28-DAS-treated virus-infected cells were 23% of untreated and infected cells. The synthesis of thymidine kinase (TK) was reduced by 28-DAS at early stage. In conclusion, 28-DAS inhibited the replication of HSV-1, reduced the synthesis of HSV-1 TK, and led to the formation of defective nucleocapsids.

Influenza A virus infection modulates the expression of type IV collagenase in epithelial cells.

We investigated the effect of influenza A/Beijing/353/89 (H3N2) virus infection on the expression of type IV collagenase in two different types of epithelial cell. Depending on the cell line infected, the viral infection caused changes in the expression of type IV collagenase. The expression of matrix metalloproteinase-9 (MMP-9; 92 kDa) but not of matrix metalloproteinase-2 (MMP-2; 72 kDa) was stimulated in Vero cells. In MDCK cells, the MMP-2 production increased with the virus infection. According to the enzymatic activity revealed with zymography, the MMP-9 promoter activity rose by a factor of over 1788 in influenza A virus-infected Vero cells but not in MDCK cells. The tissue inhibitor of metalloproteinase, TIMP-1, had increased slightly (2.3-fold) in Vero cells 48 hours after the infection, but in MDCK cells, influenza A virus had no effect on the TIMP-1 expression. In conclusion, the MMP-9 and -2 expression by influenza A virus infection are modulated at transcriptional level, depending on the epithelial cell line.

Identification of mycobacterial species by comparative sequence analysis of the RNA polymerase gene (rpoB).

For the differentiation and identification of mycobacterial species, the rpoB gene, encoding the beta subunit of RNA polymerase, was investigated. rpoB DNAs (342 bp) were amplified from 44 reference strains of mycobacteria and clinical isolates (107 strains) by PCR. The nucleotide sequences were directly determined (306 bp) and aligned by using the multiple alignment algorithm in the MegAlign package (DNASTAR) and the MEGA program. A phylogenetic tree was constructed by the neighbor-joining method. Comparative sequence analysis of rpoB DNAs provided the basis for species differentiation within the genus Mycobacterium. Slowly and rapidly growing groups of mycobacteria were clearly separated, and each mycobacterial species was differentiated as a distinct entity in the phylogenetic tree. Pathogenic Mycobacterium kansasii was easily differentiated from nonpathogenic M. gastri; this differentiation cannot be achieved by using 16S rRNA gene (rDNA) sequences. By being grouped into species-specific clusters with low-level sequence divergence among strains of the same species, all of the clinical isolates could be easily identified. These results suggest that comparative sequence analysis of amplified rpoB DNAs can be used efficiently to identify clinical isolates of mycobacteria in parallel with traditional culture methods and as a supplement to 16S rDNA gene analysis. Furthermore, in the case of M. tuberculosis, rifampin resistance can be simultaneously determined.

Over-expression of urokinase receptor in human epidermoid-carcinoma cell line (HEp3) increases tumorigenicity on chorio-allantoic membrane and in severe-combined-immunodeficient mice.

Using chorio-allantoic membranes (CAMs) of chick embryos and severe-combined-immunodeficient (SCID) mice, we investigated the effects of urokinase-type plasminogen-activator receptor (u-PAR) over-expression on the process of invasion and tumorigenicity. By the transfection of u-PAR cDNA, 3 u-PAR-over-expressing clones expressing 1.6- to 4.6-fold more u-PAR mRNA than parent cells were obtained from a human epidermoid-carcinoma cell line, HEp3, that expresses urokinase-type plasminogen activator (u-PA) and u-PAR. All the u-PAR-over-expressing clones showed greater invasiveness (13 to 29%) than that of parent HEp3 cells on CAMs. Immunohistochemistry revealed densely stained u-PAR-positive cells near the margin of the tumor, where a u-PAR-over-expressing clone, designated SM-3, was invading thickened fibrous tissue on CAMs. Three u-PAR-overexpressing clones formed larger tumors (>40 mm3) than did parent HEp3 cells on CAMs. Moreover, when the u-PAR-overexpressing clone (SM-3) was injected s.c. into the back of the SCID mice it produced a larger tumor volume than the control (HEp3) and down-regulated (AS-2) clones and significantly shortened the survival of SCID mice. These results demonstrate that increased u-PAR expression is an important factor in determining the malignant phenotype that makes cancer cells more invasive and tumorigenic.