BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) has a heteroresistant nature, so methicillin resistance is influenced by various culture conditions, such as temperature, incubation time, and NaCl content in the medium. Mueller Hinton (MH) agar containing 2% NaCl and mannitol salt agar (MSA) with oxacillin disk were evaluated for the detection of methicillin resistance. METHODS: Disk diffusion test on plain Mueller- Hinton (MH) agar, 2% NaCl MH agar, and MSA with 1 microgram oxacillin disk was performed in 70 Stap hylococcus aureus isolates. Oxacillin MIC was determined by E-test. As a gold standard of methicillin resistance, mecA gene was amplified by PCR and detected by agarose gel electrophoresis. RESULTS: Plain MH agar could not detect heterogeneous resistance in 12 S. aureus isolates (18%), but 2% NaCl MH agar and MSA could correctly detect homogeneous and heterogeneous resistance. S. aureus isolates from stool have as much as 48% heterogeneous resistance, while those from non-stool specimen have 5%. CONCLUSION: 2% NaCl and MSA can be used reliably for accurate susceptibility testing of methicillin resistance in routine laboratory.
Agar* ; Diffusion ; Electrophoresis, Agar Gel ; Mannitol* ; Methicillin Resistance* ; Methicillin* ; Methicillin-Resistant Staphylococcus aureus ; Oxacillin ; Polymerase Chain Reaction ; Staphylococcus aureus* ; Staphylococcus*

Agar-gel diffusion test (AGD), counterimmunoelectrophoresis (CIEP) and enzyme-linked immunosorbent assay(ELISA) were examined with the sera of skin test positives for paragonimiasis. The crude antigen(Paragonimus whole worm extracts: protein concentration, 7.56mg/ml) and human sera were used in AGD and CIEP. And in ELISA test, diluted antigen with 1:40,000 of crude antigen and diluted sera with 1:100, 1:200 were used in the test. The positive identical ratio between AGD and CIEP reactions is 98 percent and negative identical ratio is 100 percent. One or three precipitin bands are observed in AGD. One to seven precipitin bands are also revealed in CIEP. Especially, deeply stained bands are observed in CIEP than those of AGD. The positive identical ratios between AGD and ELISA tests are 96 percent in 1:100 diluted sera, and 94 percent in 1:200 diluted sera. But the negative identical ratios between AGD and ELISA tests are 97 percent and 99 percent respectively in 1:100 and 1:200 diluted sera. The positive identical ratios between CIEP and ELISA tests are 98 percent and 96 percent respectively in 1:100 and 1:200 diluted sera, but also 97 percent and 99 percent in 1:100 and 1:200. Control sera, such as clonorchiasis, amoebiasis and toxoplasmosis, revealed all negatives with Paragonimus antigen in AGD, CIEP and ELISA tests. By above results, ELISA was most sensitive, next CIEP and AGD. But AGD test appears to be more useful when used to crude antigen without cross reaction with other parasitic infections. CIEP test is basically equal in terms of precipitin reaction, but CIEP is able to be detected more sensitively and rapidly though less simple in handiwork than AGD. Consequently, three methods for immunological tests of paragonimiasis have good correlations with one another. Also, each of these has both merits and demerits in immunological test for paragonimiasis. But the ELISA test was proved to be the most sensitive and convenient tool for mass screening test, especially in case of using purified antigen.
parasitology-helminth-trematoda ; Clonorchis sinensis ; clonorchiasis ; ELISA ; immunology ; diagnosis ; paragonimiasis ; Paragonimus westermani ; agar-gel diffusion ; counterimmunoelectrophoresis

BACKGROUND: The importance of extended-spectrum beta-lactamases (ESBL) produced in gramnegative bacilli is now well recognized, but most clinical laboratories have problems in detecting and interpreting ESBL and implicating the findings in nosocomial infections caused by ESBL producing gram-negative bacilli. The present study aims primarily to evaluate the distributions of these enzymes among Escherichia coli and Klebsiella pneumoniae, the most frequent isolates of Enterobacteriaceae producing ESBL, to differentiate the types of enzymes in theses isolates and finally to relate the clonality of specific types within a part of Daegu city. METHODS: The clinical isolates of 1, 242 E. coli and 859 K. pneumoniae were screened for ESBL production by the disk diffusion method of the National Committee of Clinical Laboratory Standard, and it was confirmed by the double-disk synergy test (DDS). Antimicrobial susceptibility test was performed by the agar dilution method. The presence of -lactamase was tested by polymerase chain reaction (PCR) and plasmid analysis. Isoelectric focusing and nucleotide sequence analysis were performed to evaluate ESBL types. Pulsed-field gel electrophoresis (PFGE) of XbaI-digested DNA fragments was carried out to determine the extend of clonality within the hospital. RESULTS: Of 34 isolates of E. coli and 31 isolates of K. pneumoniae ramdomly selected from those isolates screened for ESBL production were further tested by DDS to confirm its production: 30 (88.2%) E. coli and 29 (93.5%) K. pneumoniae were positive. TEM-52 and SHV-12 were present both in E. coli and K. pneumoniae, but SHV-2a was distributed only in K. pneumoniae. The resistance was transferable in 66.7% of E. coli and 68.9% of K. pneumoniae. Six and 5 PFGE patterns were shown by E. coli and K. pneumoniae, respectively. Among the 5 patterns of K. pneumoniae, type B was dominant, suggesting a clonal outbreak in the hospital. CONCLUSIONS: The ESBL specific enzyme types were TEM-52, SHV-2a and SHV-12. Despite many different PFGE patterns of the ESBL producing isolates, a few outbreak and edemic clones appear to be prevalent in Dongsan Medical Center.
Agar ; Base Sequence ; beta-Lactamases* ; Clone Cells ; Cross Infection ; Daegu ; Diffusion ; DNA ; Electrophoresis, Gel, Pulsed-Field ; Enterobacteriaceae ; Escherichia coli* ; Isoelectric Focusing ; Klebsiella pneumoniae* ; Plasmids ; Pneumonia ; Polymerase Chain Reaction

Precipitin reaction between host organ extracts and antisera for Paragonimus and Clonorchis were studied to know whether the antigens from two species of trematodes have common characters with those of host organs. The brain, lung, heart, liver and muscle of rat and rabbit were extracted in 0.1 percent saline solution by freezing-thawing technique. For immunization of rabbit, the extracts of Paragonimus and Clonorchis was emulsified with equal amount of Freund's adjuvant and 1.0 ml of the mixture was injected subcutaneously once every 10 days for 5 times, and 3 days after the last immunization, antisera were prepared. Precipitin bands by agar-gel double diffusion methods were examined between organ extracts of rabbit or rat and Paragonimus or Clonorchis antisera. The results could be summarized as follows: Precipitin bands were appeared between extracts of liver, heart and muscle of rat and anti-Clonorchis rabbit sera but not appeared against brain and lung. Precipitin bands were appeared between extracts of lung, muscle and liver of rat and anti-Paragonimus rabbit sera but not appeared against brain and heart. Precipitin bands were appeared between extracts of liver and muscle of rabbit and anti-Clonorchis rabbit sera but not appeared against brain, heart and lung. Precipitin bands were appeared between extracts of lung and muscle of rabbit and anti-Paragonimus rabbit sera but not appeared against liver. The extracts of Paragonimus westermani showed precipitin bands against liver and brain of rat and liver of rabbit in agar-gel double diffusion.
parasitology-helminth-trematoda ; Paragonimus westermani ; Clonorchis sinensis ; immunolgy ; brain-lung-heart-liver-muscle ; rabbit ; rat ; agar-gel double diffusion ; Freund's adjuvant

BACKGROUND: Increasing numbers of Acinetobacter spp. resistant to multiple drugs, including carbapenem, has been a serious problem. The aims of this study were to determine carbapenem resistance patterns and mechanisms, as well as to study the molecular epidemiology of Acinetobacter spp. METHODS: Clinical isolates of Acinetobacter spp. were collected from May to November in 2006. Antimicrobial susceptibility testing was performed using CLSI disk diffusion and agar dilution methods. Metallo-beta-lactamase- and OXA carbapenemase-producing isolates were detected by PCR. Carbapenem resistance and hydrolytic activities were compared according to OXA type and presence of ISAba1. Pulsed-field gel electrophoresis (PFGE) was performed to determine the epidemiologic features. RESULTS: The imipenem non-susceptible rates were variable from 10% to 67%. Among 151 isolates carrying bla(OXA-51-like), 75 isolates carried both bla(OXA-51-like) and ISAba1, and 25 isolates had both bla(OXA-51-like), bla(OXA-23-like), and ISAba1. Carbapenem MICs of both bla(OXA-51-like) and ISAba1-carrying isolates were higher than those with bla(OXA-51-like) only. Carbapenem MICs of bla(OXA-23-like)-carrying isolates were higher than those with both bla(OXA-51-like) and ISAba1. Both bla(OXA-51-like) and ISAba1-carrying isolates and blaOXA-51-like, blaOXA-23-like, and ISAba1-carrying isolates demonstrated higher hydrolysis activities in oxacillin and carbapenems. Most of the tested isolates were susceptible to tigecycline, and all of them were susceptible to colistin. Pulsed-field gel electrophoresis suggested that there had been several outbreaks of bla(OXA-23-like) and bla(OXA-51-like)-positive strains. CONCLUSION: Carbapenem non-susceptible Acinetobacter isolates and OXA carbapenemase-producing isolates were prevalent. Dissemination of bla(OXA)-harboring isolates may make it difficult to treat infections due to carbapenem-resistant Acinetobacter spp. Further surveillance studies are required to prevent the spread of carbapenem resistance.
Acinetobacter ; Agar ; Carbapenems ; Colistin ; Diffusion ; Disease Outbreaks ; Electrophoresis, Gel, Pulsed-Field ; Hydrolysis ; Imipenem ; Lifting ; Minocycline ; Molecular Epidemiology ; Oxacillin ; Oxytocin ; Polymerase Chain Reaction

BACKGROUND: Enterococcal infections have become extremely difficult to manage because of an increase in antibiotic resistance among enterococci. In Europe, the use of avoparcin in animals was reported to be the cause of vancomycin-resistant enterococci (VRE) transmission to humans. In this study, we performed antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE) to characterize the genetic relatedness of VRE of human and chicken. METHODS: Ninety strains of VRE were isolated from clinical specimens in three University hospitals located in Seoul and Kyungi province in 2001-2002. Thirty isolates of VRE were collected from four chicken farms located in areas remotely distanced from each other. The isolates were identified to the species level by conventional biochemical tests and commercial kits. Antimicrobial susceptibilities were tested by the NCCLS disk diffusion and agar dilution methods. For a molecular epidemiologic analysis, PFGE was performed. RESULTS: Among the 90 clinical isolates were 73 vancomycin-resistant Enterococcus faecium (VREFM) and 17 vancomycin-resistant E. faecalis (VREFA). The resistant rates of VREFA to ampicillin, levofloxacin and tetracycline were 0%, 100%, and 100%, respectively, and for VREFM, 100%, 96%, and 26%, respectively. However, the resistant rates of VREFM isolated from chicken were 19% to ampicillin, 0% to levofloxacin, and 100% to tetracycline. The PFGE patterns of genomic DNA of the clinical isolates were very diverse, suggesting a polyclonal spread of VRE, although some isolates had an identical PFGE pattern, indicating a mini-outbreak due to a clonal spread. The PFGE patterns of genomic DNA of the chicken isolates were very different from those of the human isolates. CONCLUSIONS: VRE isolates from human and chicken showed very different antimicrobial susceptibilities and PFGE patterns. These results suggest that VRE isolated from human and chicken are not closely related genetically.
Agar ; Ampicillin ; Animals ; Chickens* ; Diffusion ; DNA ; Drug Resistance, Microbial ; Electrophoresis, Gel, Pulsed-Field ; Enterococcus faecium ; Enterococcus* ; Epidemiology ; Europe ; Hospitals, University ; Humans ; Levofloxacin ; Seoul ; Tetracycline ; Vancomycin

BACKGROUND: Vancomycin-resistant enterococci (VRE) with vanA gene have been reported as a significant nosocomial pathogen. The vanA gene cluster (Tn1546) located on mobile DNA elements is known to be transferable from VRE to other enterococci. The purpose of this study was to investigate the genetic relationship between the vanA VRE strains isolated from hospitalizd patients and poultry. METHODS: Total 145 isolates, including 58 E. faecium, 12 E. faecalis, 3 E. casseliflavus, and 4 E. gallinarum from humans and 68 E. faecium from poultry, were studied. Antimicrobial susceptibility tests were done by disk diffusion or agar dilution methods and molecular epidemiological analysis was performed by pulsed-field gel electrophoresis (PFGE). The internal and structural regions of vanA gene cluster were analyzed by PCR fragment length polymorphism, restriction enzyme, and sequencing of Orf2D region and vanXY intergenic region. The point mutation at Tn1546 nucleotide position 8234 (G->T) within the vanX gene was screened with DdeI restriction enzyme. RESULTS: The antibiotic resistance patterns of human isolates were different from those of poultry. PFGE patterns revealed high heterogeneity. Three PCR fragment length patterns in the vanA gene cluster were found : (I) PCR amplicon of the same size as prototype (E. faecium BM4147) in 17% of human isolates and 100% of poultry ones; (II) PCR amplicon for vanXY intergenic region due to an insertion between vanX and vanY genes in 2.5% of human isolates; (III) the insertions in vanX-vanY intergenic and Orf2 regions in 81% of human isolates. The T type in vanX gene of human and poultry isolates was not found. CONCLUSION: Despite the diverse PFGE patterns, 81% of human and all of poultry isolates belonged to vanA gene cluster type III and I, respectively. These results indicate that the horizontal spread of vanA gene is occurring among genetically diverse strains of VRE in Korea.
Agar ; Diffusion ; DNA ; DNA, Intergenic ; Drug Resistance, Microbial ; Electrophoresis, Gel, Pulsed-Field ; Humans ; Korea* ; Multigene Family ; Point Mutation ; Polymerase Chain Reaction ; Population Characteristics ; Poultry*

BACKGROUND: Recently, an acquired resistance to vancomycin in enterococci has become a serious clinical problem. For the prevention of further propagation of vancomycin-resistant enterococci (VRE), epidemiological study of the infection is essential, but studies on the VRE infection are rare in Korea. We conducted an analysis of the epidemiology of a VRE outbreak in a neonatal intensive care unit (NICU) to clear up the route of propagation of the VRE. METHODS: Vancomycin-resistant Enterococcus faecium (VREFM) strains were isolated from urine specimens of seven patients, rectal swabs from seven patients, and three skin swabs from two patients in the Kosin Medical Center neonatal intensive care unit, Pusan, Korea. Antimicrobial susceptibilities were tested by a disk diffusion method and agar dilution method. Genotypes of vancomycin-resistance were determined by PCR and SmaI-digested genomic enterococcal DNAs were analyzed by pulsed-field gel electrophoresis. RESULTS: All of the 17 strains of VREFM were resistant to ampicillin, vancomycin, and teicoplanin and they showed the same genotype (vanA). SmaI-digested genomic DNAs of seven strains isolated from urine were typed as I (1), II (1), IIIb (4), and IV (1). Three strains from skin swabs were I (2) and II (1). Six strains from rectal swabs were I (2), II (1), and IIIa (3). Genomic DNA typing of one isolate from a rectal swab failed. Each genomic DNA type of VREFM strains isolated from skin swabs of two patients were the same with those from urine specimens as I and II, respectively. CONCLUSIONS: This study suggests that VRE strains colonized in the intestines can cause infections after skin colonizing and can be transmitted/propagated to other people through skin contact. In conclusion, it is important for the prevention of the dissemination of VRE that controls for patients' skin hygiene, as well as hand washing by medical persons, be put in place.
Agar ; Ampicillin ; Busan ; Colon ; Diffusion ; DNA ; DNA Fingerprinting ; Electrophoresis, Gel, Pulsed-Field ; Enterococcus faecium ; Epidemiologic Studies ; Epidemiology ; Genotype ; Hand Disinfection ; Humans ; Hygiene ; Infant, Newborn ; Intensive Care, Neonatal* ; Intestines ; Korea ; Molecular Epidemiology* ; Polymerase Chain Reaction ; Skin ; Teicoplanin ; Vancomycin

BACKGROUND: Pathogenic fungi infect humans, especially immunocompromised patients, with superficial or deeply invasive pattern. In the past 20 years, fungal infections have been increased dramatically resulted by increment of organ transplantation, cancer, AIDS patients, or use of broad-spectrum antibacterial agents. Fungal infections are now important causes of morbidity and mortality of hospitalized patients. but there is no effective antifungal antibiotics as well as antibacterial antibiotics OBJECTIVE: Effective new antifungal antibiotics are needed for the treatment of mycosis. So in an effort to develop effective antifungal antibiotics, we screened over 600 isolates of Streptomyces sp. from soil. METHODS: Antifungal producing strain was selected using disk diffusion method, An antifungal substance (AF1) was purified with ethyl acetate extraction, silica gel column chromatography and reverse phase HPLC. MICs of AF1 were detected by agar dilition method. RESULTS: The compound showed UV maxima of 307, 321, 340, 359 nm indicating methylpentaene. Minimum inhibitory concentrations of the AF1 were 3.7 microgram/ml against mold, and 3.7 - 7.4 microgram/ml against Candida species. AFI was also active against Crytococcus neoformans, with MIC of 0.9 microgram/ml. The concentration of AF1 for K+ ion release from human red blood cell and hemolysis were 5 microgram/ml. CONCLUSION: The antibiotic purified from culture broth of Streptomyces sp. WCM-9 was a polyene antifungal antibiotic which have broad spectrum antifungal activity.
Agar ; Anti-Bacterial Agents ; Antifungal Agents ; Candida ; Chromatography ; Chromatography, High Pressure Liquid ; Diffusion ; Erythrocytes ; Fungi ; Hemolysis ; Humans ; Immunocompromised Host ; Microbial Sensitivity Tests ; Mortality ; Organ Transplantation ; Silica Gel ; Soil ; Streptomyces* ; Transplants

BACKGROUND: Pseudomonas aeruginosa is a major cause of nosocomial infections. During recent three years, the isolation rate was high in Wonkwang University Hospital, especially in intensive care unit(ICU) and wards of neurosurgery. We performed this study to investigate the isolation rate and mode of transmission of P. aeruginosa, and usefulness of antibiotic resistance, colonial characteristics and PFGE patterns in the epidemiologic survey. METHOD: From Aug. 1996 to Oct. 1998, 1,682 strains of P. aeruginosa were isolated. For the isolation of P. aeruginosa, environment and 18 nurses's hands were cultured. Antibiotic resistance were tested by NCCLS disk diffusion method for 298 selected strains. Among them 98 strains were evaluated for colonial characteristics(color and margin) on the blood agar and PFGE patterns restricted by Spe I, were evaluated. RESULTS: Overall isolation rate was 12% and was high in medical ICU (23%) and neurosurgical ICU(12%). The majority of specimens where P. aeruginosa was isolated were sputum 47%, urine 24% and wound 21% in decreasing order. The wards where isolation rate was high, had more resistant strains. Ninty eight strains could be classified into 39 different groups by PFGE patterns. But 29 strains belonged to five major patterns (P1-5) and were suspected as epidemic or cross-infected strains. Majority of these strains revealed resistant to two or more antibiotics and colonial phenotype of G2R, GIR(P1), G1I, G3I(P2), Wm(P3), G3I, G3R(P4) and G3I(P5) types. Forty four strains isolated from specimens(sputum, urine, wound, and stool) of 7 patients during hospitalization, revealed single or two PFGE patterns per patient. Conclusion: Transmission mode of P. aeruginosa was suspected to be patient's own GI-tract and cross contamination, especially via health care persons. Combined phenotypes of antibiotic resistance and colonial characteristics correlated well with PFGE patterns. So, in the early period of outbreak of P. aeruginosa, careful examination of colonial characteristics and antibiotic resistance patterns gave meaningful information.
Agar ; Anti-Bacterial Agents ; Cross Infection ; Delivery of Health Care ; Diffusion ; Drug Resistance, Microbial* ; Electrophoresis, Gel, Pulsed-Field ; Epidemiologic Studies* ; Hand ; Hospitalization ; Humans ; Critical Care ; Neurosurgery ; Phenotype ; Pseudomonas aeruginosa* ; Pseudomonas* ; Sputum ; Wounds and Injuries ; Surveys and Questionnaires