Control of tuberculosis is threatened by widespread emergence of drug resistance in Mycobacterium tuberculosis. Understanding the molecular basis of resistance might lead to development of novel rapid methods for diagnosing drug resistance. Rifampin is a key component among therapeutic regimens for the tuberculosis; therefore, patients who have drug resistance do not convalesce satisfactorily. The molecular mechanism of resistance to rifampin in M. tuberulosis has been elucidated. Substitutions of a limited number of highly conserved amino acids encoded by the rpoB gene are responsible for the ""single-step"" high-level resistance of M. tuberculosis to rifampin. Currently, two genotype-based protocols allow drug test from minimally grown cultured materials: (i)mutation identification by direct sequencing of PCR-amplified material. and (ii)mutation screening by PCR-SSCP. The purpose of this study is to evaluate the usefulness of the both methods. A sample of 75 isolates of M. tuberculosis was studied, and it inculded 36 rifampin-resistant strains and 39 rifampin-sensitive strains by conventional methods. Mutaions were identified in 36 rifampin-resistant isolates but in none of 39 sensitive isolates. All mutations were clustered within a region of 23 amino acids. Both methods allow detection of rifampin resistance in 2 to 3 days and will thus help in the early management of infection by M. tuberculosis.
Amino Acids ; Drug Resistance ; Humans ; Mass Screening ; Mycobacterium tuberculosis* ; Mycobacterium* ; Rifampin ; Tuberculosis

Fecal isolates of Escherichia coli which were collected from diarrheal patients and HUS patient in Pusan National University Hospital between 1990 and 1996, were serotyped and analyzed for plasmid DNA profile, biotype, HEp-2 cell adherence ability, reactivity to eae probe and for production of verotoxins (VT). In order to ease the diagnosis of EHEC infection, a LPS- based solid phase enzyme linked immunosorbent assay was utilized to detect serological diagnosis of EHEC infection. The following results were obtained. Among 150 EPEC isolates and HUS patient's stool, 7 EHEC were found. The 7 EHEC belonged to 5 different serotypes 0157:H7, 0143:H-, 0166:H-, 0128:H2, 026:H-, and 0111:H 21 previously associated with human haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). Biochemical cheracteristic analysis indicated 7 strains were biotype 1 and was found to have siderophilins suggesting their advantagous growth in vivo. For plasmid profiles, all strains had 60 MDa plasmid and several smaller sizes of plasmids. Three strains of Escherichia coli serotype 0157:H7, 0128:H2, and 026:H- showed one pattern of adherence in the HEp-2 cell assay namely, localized adherence and were positive for eae probe when tested by colony blot hybridization assay. PCR using specific primers for VT1, VT2 was tested, and all 7 strains carried VT1 gene only. PCR products of 130-bp (VT1) and 346-bp (VT2) were successfully amplified simultaneously in a single reaction. The multiplex PCR method can be used to specifically identify EHEC. The serum obtained from HUS patient of enterohemorrhagic E. coli were analyzed for rises in titer of intibody to somatic 02, 026, 0111, 0128, 0143, 0145, 0157, and 0165. Although response to the somatic 0 correlated significantly with response to the 026 rises of antibody titer to somatic 0 in acute stage of disease and anti-VT had not so many relation to that of VT. These results suggest that ELISA can be used to detect somatic 0 in serum and it is a useful method to diagnose the infection caused by EHEC rapidly.
Antibodies* ; Busan ; Colitis ; Diagnosis* ; DNA ; Enterohemorrhagic Escherichia coli* ; Enteropathogenic Escherichia coli ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Humans ; Multiplex Polymerase Chain Reaction ; Plasmids ; Polymerase Chain Reaction* ; Shiga Toxins ; Transferrin

Among the fifty-three clinical isolates of Haemophilus influenzae, nineteen isolates including eight isolates of each biotype I-VIII, six of serotype b (Hib) strains and five of nontypeable strains were characterized by SDS-PAGE about outer membrane protein (OMP), restriction enzyme analysis (REA) and rRNA gene restriction pattems. OMP patterns showed to common band patterns in each H. influenzae isolate. Based on the two major proteins, 31KDa-38KDa, isolated strains were classified into 7 subtypes. In the OMP patterns about biotype and serotype, the specific pattern of each biotype was not distinguishable, but all of the serotype b strains were shown identical unique pattern, therefore it made distinctive difference with nontypeable strains. The digested genomic DNAs with EcoRI were identical result with rRNA gene restriction. It was more subdivided into 10 ribotypes. The most common ribotype I and serotype 1 accounted for 6 strains (31.6%) and 7 strains (36.8%) of the 19 clinical isolates, respectively. Hib isolates that were both OMP subtype 1 and ribotype I accounted for 2 strains (10.5%). In the epidemiologically unrelated strains, the putative association between the subtypes could not be confirmed. According to these results, the three methods were discriminatory and appropriate techniques for epidemiological studies of H. influenzae.
DNA ; Electrophoresis, Polyacrylamide Gel* ; Genes, rRNA* ; Haemophilus influenzae type b ; Haemophilus influenzae* ; Haemophilus* ; Influenza, Human ; Membrane Proteins ; Restriction Mapping* ; Ribotyping

Eighty-two strains of Escherichia coli isolated from urine specimens in Pusan University Hospital, were serotyped and analyzed for plasmid DNA profiles, PFGE profiles, MRHA of human blood cells, HEp-2 cell adherence ability and reactivity to bfpA, LT, STh and STp DNA probes. The following results were obtained. Fifty-three of the eighty-two strains belonged to thirteen different 0 serotypes, twenty-nine strains could not be typed with the antisera used. Thirty strains (43.9%) were hemolysin producer. MRHA is present on twenty-nine strains (35.37%) of eighty-two strains. MRHA positive strains carry a plasmid of 60MDa, a putative factor involved in adherence. This plasmid might be specific for MRHA positive strains. MRHA positive strains were observed in serotype 01, 018, 055, 086a, 0119, 0126, and 0142. Twenty-six strains of E. coli showed three patterns of adherence to HEp-2 cells namely, localized, diffuse, and aggregative adhesion. Twenty-two strains hybridized with the bfpA probe, while all eighty-two strains did not hybridize with the probes, LT, STh, STp. The restriction fragment patterns of chromosomal DNA digested with AotI analysed by PFGE of hemolysin-producing E. coli ten strains were compared with eight different types. Three of E. coli serotype 01, 08 and 0126 showed the same chromosomal DNA fragment patterns.
Blood Cells ; Busan ; DNA ; DNA Probes ; Escherichia coli* ; Escherichia* ; Humans ; Immune Sera ; Plasmids ; Serotyping*

The antimicrobial agents reduced infectious diseases significantly. However, antibiotic resistance has followed for almost every antimicrobial agent. Especially, Staphylococcus aureus was one of the most notorious for the multidrug resistance. Streptomyces sp. 681 has been selected for antibiotic-producing strain against methicillin-resistant Staphylococcus aureus (MRSA) from 1,000 strains of Actinomycetales which had been isolated from soil. In antimicrobial susceptibility test, all of the test strains were susceptible to vancomycin. However, most strains of Staphylococcus aureus were found to be resistant to methicillin. Ninety eight (75%) strains out of 129 strains showed multiple resistance pattern to more than 5 antimicrobial agents. The MIC values of the purified antibiotic (K-681) were 1-32 ug/ml against Gram-positive bacteria compared to >128 ug/ml against Grarn-negative bacteria or fungi. The MIC was 8 ug/ml for 90% of the 129 clinical isolates of S. aureus. The antibiotic showed no cytotoxicity against P 388, HeLa, and S180 at the concentration of 500 ug/ml.
Actinomycetales ; Anti-Infective Agents ; Bacteria ; Communicable Diseases ; Drug Resistance, Microbial ; Drug Resistance, Multiple ; Fungi ; Gram-Positive Bacteria ; Methicillin ; Methicillin-Resistant Staphylococcus aureus ; Soil ; Staphylococcus aureus* ; Staphylococcus* ; Streptomyces* ; Vancomycin

Helicobacter pylori is a causative agent of type B gastritis and plays a central role in the pathogenesis of gastroduodenal ulcers and gastric cancer. Strategies for the control of H. pylori- induced gastroduodenal diseases based on conventional measures are still of limited utility. Therefore, it seems worthwhile to make a break-through as an alternative strategy by reviewing the host-parasite relationship of H. pylori infection on the basis of genomic structure. In this study, we tried to construct a physical map of H. pylori genome. Chromosomal DNA from a Korean prototype strain, H. pylori 51 was digested with 42 restriction endonucleases to identify restriction patterns suitable for mapping the genome. We identified three enzymes, ApaI, NotI and Sfil, which gave a small number of DNA fragments of higher molecular weight that were well resolved after pulsed-field gel electrophoresis. The H. pylori chromosome contained 7 ApaI fragments ranging from 167 to 311 kb, 7 NotI fragments ranging from 5 to 516 kb and 2 SfiI fragments of 332 and 1,347 kb in size. The genome size of the strain is 1,679 kb. A circular physical map of the H. pylori chromosome was constructed by aligning 3 kinds of restriction fragments by Southern blot analysis of simple ApaI, NotI and SfiI digests or double NotI/ApaI and NotI/SfiI digests with the various probes. When the physical map of H. pylori strain 51 compared with that of strain 26695 of which the cornplete genome sequence was reported, completely different restriction patterns were shown, which suggests the genomic diversity in H. pylori.
Blotting, Southern ; DNA ; DNA Restriction Enzymes ; Electrophoresis, Gel, Pulsed-Field ; Gastritis ; Genome ; Genome Size ; Helicobacter pylori* ; Helicobacter* ; Host-Parasite Interactions ; Molecular Weight ; Peptic Ulcer ; Stomach Neoplasms

Clostridium perfringens is an anaerobe responsible for a wide range of diseases in animals and humans. Symptoms associated with C. perfringens food poisoning are caused by enterotoxin expressed only during sporulation of C. perfringens. It has been known that only 6% of global C. perfringens isolates carry the enterotoxin gene. We found 2 strains of enterotoxigenic C. perfringens out of 33 strains isolated from various sources in Korea using PCR. It was also found that these two strains were both type A that were strongly associated with food poisoning by checking the presence of four major lethal toxins (a-, B-, e-, l-toxin) using PCR. These results suggest that foodborne illness caused by C. perfringens may be common in Korea and that public education is necessary to prevent contamination of foods by this organism.
Animals ; Clostridium perfringens* ; Clostridium* ; Education ; Enterotoxins* ; Foodborne Diseases ; Humans ; Korea* ; Polymerase Chain Reaction

Prospect Hill virus (PHV) is related antigenically but distinct to Hantaan virus. As a member of genus Hantavirus, PHV has three segmented RNA genome. Among these segments, Small segment(S) encodes nucleocapsid protein (NP) as structural protein and also may do functional nonstructural protein(NSs). We performed in vitro transcription to produce vRNA of PHV S genome. For the first step of in vitro transcription of S genome of PHV, the S RNA segrnent which is 1,675 nucleotides long was amplified by RT-PCR using PCR primers built according to cDNA sequence of PHV S genome. Next, a new PCR primer appended above downstream primer to T7 phage promoter sequence was reconstructed to obtain PCR product containing T7 promoter. Then another PCR was performed. Using this PCR product as the template, in vitro run-off transcription without cloning by transcriptional vector was performed to obtain viral- sense RNA transcript. Thereafter, the size of transcript was assessed by running on formaldehyde agarose gels. Since the transcription reactants contain a-S UTP, the transcript is detectable by autoradiography. The transcript was also detectable by northern hybridization with a-P dCTP- labelled PHV amplicon probe (319 bp) and the initiation start point of run-off transcription was also determined by primer extention analysis. Our data indicate that the in vitro transcript could be produced from the PCR product amplified by PCR primer containing T7 phage promoter without cloning into a phage transcription vector.
Autoradiography ; Bacteriophage T7 ; Bacteriophages ; Clone Cells ; Cloning, Organism ; DNA, Complementary ; Formaldehyde ; Gels ; Genome* ; Hantaan virus ; Hantavirus ; Nucleocapsid Proteins ; Nucleotides ; Polymerase Chain Reaction ; RNA ; Running ; Sepharose ; Uridine Triphosphate ; Viruses*

Hantaviruses are members of the family Bunyaviridae, the etiologic agents of Hemorrhagic fever with renal syndrome (HFRS) and Hantavirus Pulmonary Syndrome (HPS). They are negative-sense, single-stranded RNA viruses possessing a large (L), medium (M) and small (S) genomic segment which encodes a viral polymerase, envelope glycoproteins (G1 and G2) and a nucleocapsid (N) protein, respectively. Seoul (SEO) virus, the causative agent of clinically mild HFRS in worldwide, was isolated from lung tissues of urban rat (Rattus norvegicus) captured in Seoul, Korea, 1982. To clarify the antigenic characteristics and the differentiation of serotypes of hantavirus, 8 hybridoma cell lines secreting monoclonal antibodies (MAbs) against SEOV 80-39 strain were produced by fusion of SP2/0-Ag14 mouse myeloma cells with spleen cells of BALB/c mice, immunized with SEOV. Reactivities of these MAbs were examined by the indirect immunofluorescence assay (IFA), enzyme linked immunosorbent assay (ELISA), immunoblot, high density particle agglutination (HDPA) and plaque reduction neutralization test (PRNT). Eight out of 235 hybridomas secreted MAbs of Seoul virus 80-39 continuously, and these eight MAbs were all IgG. The isotypes of these 8 MAbs are; one clone (F3-3C) was IgG1, six (F1-1B9B, F1-3B, F1-3D, F4-1E, F4-3F, F4- 6C) were IgG2a and one (F1-1B9F) was IgG2b. Seven MAbs (F1-1B9B, F1-1B9F, F1-3B, F1- 3D, F3-3C, F4-1E, F4-3F) reacted with nucleocapsid protein (M.W. 50K) of SEOV by immunoblot. All eight MAbs were cross-reacted with Hantaan (HTN) virus, one (F4-3F) was cross-reacted with Puumala (PUU) virus and two (F1-1B9B, F1-3B) were cross-reacted with Prospect Hill (PH) virus by IFAT. None of these 8 MAbs had neutralizing activity.
Agglutination ; Animals ; Antibodies, Monoclonal* ; Bunyaviridae ; Cell Line ; Clone Cells ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique, Indirect ; Glycoproteins ; Hantavirus ; Hantavirus Pulmonary Syndrome ; Hemorrhagic Fever with Renal Syndrome ; Humans ; Hybridomas ; Immunoglobulin G ; Korea ; Lung ; Mice ; Neutralization Tests ; Nucleocapsid ; Nucleocapsid Proteins ; Rats ; RNA Viruses ; Seoul virus* ; Seoul* ; Spleen

We isolated Triton X-100 solubilized protein (TSP) antigen which may be preferentially associated with the cell wall of M. tuberculosis. In this study, the proliferative activities and cytokine mRNA expression patterns of the TSP antigen were investigated in peripheral blood mononuclear cells (PBMCs) and lymph node mononuclear cells (LNMCs) from 4 patients with tuberculous lymphadenitis. The results of the TSP antigen were compared with those of the PPD antigen, known as a major seretory protein antigen of M. tuberculosis. The peak proliferative response to the TSP by PBMCs was observed at 0.1 ug/ml, whereas that of LNMCs was at 1.0 ug/ml. All of the patients showed greater blastogenic responses for the PPD than those for the TSP. IFN-r, IL-2, and IL-2Ru mRNA production from PBMCs after stimulation with the TSP were greatly augmented after 48 hrs, whereas IL-4 and IL-10 mRNA were gradually suppressed. In addition, high levels of IL-12 p40 mRNA were detected by PBMCs to the TSP antigen at 3 hrs. Elevated IFN-r and IL-2 mRNA production were observed in freshly isolated LNMCs, whereas IL-4 mRNA production was undetectable in either freshly isolated or mycobacterial antigen-stimulated LNMCs. Furthermore, IL-10 mRNA expression from LNMCs was markedly increased by the PPD antigen, but it was considerably reduced by the TSP antigen after 18 hrs. These data suggest that the TSP antigen may be a strong inducer of cytokine mRNA such as IFN-r, IL-2, and IL-12 which are involved in Thl cell and macrophage activation, and inhibit IL-10 mRNA production in LNMCs. In conclusion, the TSP antigen can be used as a preferential Thl cell immunogen in tuberculous lymphadenitis.
Cell Wall ; Humans ; Interleukin-10 ; Interleukin-12 ; Interleukin-2 ; Interleukin-4 ; Lymph Nodes* ; Macrophage Activation ; Mycobacterium tuberculosis* ; Mycobacterium* ; Octoxynol ; RNA, Messenger* ; Tuberculosis ; Tuberculosis, Lymph Node*