The feasibility of flow cytometric antifungal susceptibility testing has been studied using the fluorescent anionic membrane potential probe, bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]. The in vitro antifungal susceptibility testing of amphotericin B was performed on 8 Candida isolates from clinical specimens and 2 ATCC strains by flow cytometry with the results compared to those of the National Committee of Clinical Laboratory Standards (NCCLS) M27-T, broth macrodilution method. The flow cytometric method is based on an increase of fluorescence given out by DiBAC4(3) in fungi when they are killed by antifungal agents. Minimum inhibitory concentration (MIC) of amphotericin B ranged from 0.25 to 1 microg/mL. All results agreed within +/-2 dilution between the flow cytometric method and the M27-T method. MIC with ATCC strains were within recommended ranges of M27-T. The new flow cytometric method revealed a clear and distinct reproducible test end point. A four hr of incubation was sufficient for the test. In conclusion, flow cytometry using DiBAC4(3) is a rapid and accurate in vitro antifungal susceptibility testing method.
Amphotericin B/pharmacology* ; Barbiturates ; Candida/drug effects* ; Flow Cytometry/methods* ; Fluorescent Dyes ; Human ; Isoxazoles ; Microbial Sensitivity Tests

BACKGROUND: Vancomycin-resistant enterococci (VRE) have been increasingly isolated world-wide as a nosocomial pathogen. To target infection control, epidemiologic investigations of VRE should include analysis of the resistance gene in addition to typing of strains. We performed molec-ular characterization of the vanA resistance gene to evaluate the inter or intraconstitutional spread. METHODS: Twenty isolates of VanA VRE from the Centers for Disease Control and Prevention (CDC) and 17 from Ajou University Hospital (AUH) were investigated. Minimum inhibitory concen-trations of vancomycin, teicoplanin, and ampicillin were tested by the agar dilution method. Pulsed-field gel electrophoresis (PFGE) and long PCR-restriction fragment length polymorphism (long PCR-RFLP) were performed. The long PCR negative strains were typed by ORF1-, vanS-vanH-, vanX-, vanY-vanZ-, vanZ-, and IS1216-specific PCRs. Filter matings were performed by using rifampin-resistant, fusidic acid-resistant E. faecalis J H2-2 as the recipient. RESULTS: The PFGE from the VRE of the CDC showed 15 patterns including 4 clusters and PFGE from isolates of AUH revealed 6 patterns including 3 clusters. Tn1546 amplicons were detected in 18 of 20 (90%) CDC strains and 16 of 17 (94%) AUH strains. RFLP of Tn1546 amplicons revealed 5 different patterns in the VRE of the CDC strains, and 2 patterns in the VRE of the AUH strains. The mean transfer efficiency of the CDC and the AUH strains are 3.0 X 10(-8)and 4.9 X 10(-5)transconju-gant/ donor, respectively. CONCLUSIONS: Molecular typing of isolates from the CDC suggests the horizontal spread of vanA genes among genetically diverse strains. Analysis of the VRE from the AUH shows a mixed pattern with clonal dissemination of strains and horizontal transfer of vanA.
Agar ; Ampicillin ; Centers for Disease Control and Prevention (U.S.) ; Electrophoresis, Gel, Pulsed-Field ; Furosemide ; Humans ; Infection Control ; Molecular Typing ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Teicoplanin ; Tissue Donors ; Vancomycin

Since vancomycin-resistant enterococci (VRE) were first isolated in Europe, rates of VRE colonization and infection have risen steadily. Today VRE have emerged as important nosocomial pathogens worldwide; hence, it is crucial to understand the underlying mechanism in the spreading of VRE. This article reviews the mechanism of resistance to vancomycin and global epidemiology of VRE, as well as the current molecular techniques that are being applied to the epidemiological studies of VRE.
Colon ; Epidemiologic Studies ; Europe ; Vancomycin

BACKGROUND: The vanA gene cluster of vancomycin-resistant enterococci (VRE) is carried as a part of Tn1546-like elements. In this study we characterized the structure of Tn1546-like elements in Enterococcus. faecium isolated from patients in Korea. The isolates were also typed by pulsed-field gel electrophoresis (PFGE). METHODS: During 2000, 21 clinical isolates of vanA-containing E. faecium were collected from ten university hospitals in Korea. E. faecium BM4147 was used as a control. PFGE was performed on a CHEF-DR III apparatus. For structural analysis of Tn1546, the overlapping PCR amplification of internal regions of Tn1546 was performed. The purified PCR products were directly sequenced by using ABI Prism 3100 DNA SEQUENCER. RESULTS: All isolates were divided into 3 types according to the distribution of insertion sequences (IS elements) integrated Tn1546 elements. Type I and II were characterized by an IS1542 insertion in the orf2-vanR intergenic region and an IS1216V insertion in the vanX-vanY intergenic region. Type III represented two copies of IS1216V at the orf1 and in the vanX-vanY intergenic region as well as IS1542 in the orf2-vanR intergenic region. No isolates were identical to the prototype, which was identical to the predicted pattern for the published sequence of Tn1546. The PFGE results revealed that all strains except A13, C1, A2 and A9 were genetically unrelated. CONCLUSIONS: The distribution of IS in Tn1546-like elements of the Korean isolates is similar to that of the European VREs. Considering the results of PFGF and Tn1546 typing, the horizontal transfer of vanA resistance gene may be occurring among genetically diverse strains of E. faecium in Korea.
DNA ; DNA, Intergenic ; Electrophoresis, Gel, Pulsed-Field ; Enterococcus ; Hospitals, University ; Humans ; Korea ; Multigene Family ; Polymerase Chain Reaction

BACKGROUND: Nosocomial infections caused by vancomycin-resistant enterococci (VRE) are increasing problem in Korea. Until now, no nationwide study has been performed. The aim of the present study was to monitor the antimicrobial resistance of vancomycin-resistant Enterococcus faecium (VREF). METHODS: Two hundred and two E. faecium isolated in 10 teaching hospital were studied. To detect VRE, the brain heart infusion agar containing 6 /mL vancomycin was used as the screening agar. The MIC was determined using agar dilution test. The vancomycin resistance genes (vanA, vanB & vanD) and genes (aac(6 ') Ie-aph(2 ") Ia & ant(6 ') Ia encoding the aminoglycoside-modifying enzymes were detected by multiplex PCR using specific primers. RESULTS: Thirty-nine VREF were detected from 202 isolates. All had vancomycin MICs > or =256 /mL and harboured vanA gene. No isolates revealed positive results for the vanB or vanD gene. However, the MIC range for teicoplanin was 2 to > or =256 /mL. All isolates with gentamicin MIC > or = 500 /mL gave positive results for the aac(6 ') Ie aph(2 ") Ia genes and with streptomycin > or =2000 /mL gave positive results for the ant(6 ') Ia gene. CONCLUSIONS: All VREF harboured vanA gene. According to MIC tests, 7 isolates(18%) showed intermediate or susceptible to teicoplanin. Therefore we need a study concerning the clinical meaning. The VREF in Korea contain at least one of genes encoding the aminoglycoside-modifying enzymes. This means there are only limited numbers of antibiotics to choose.
Agar ; Anti-Bacterial Agents ; Brain ; Cross Infection ; Enterococcus faecium* ; Enterococcus* ; Gentamicins ; Heart ; Hospitals, Teaching ; Korea* ; Mass Screening ; Multiplex Polymerase Chain Reaction ; Streptomycin ; Teicoplanin ; Vancomycin ; Vancomycin Resistance

BACKGROUND: The Korean Society of Laboratory Medicine (KSLM) Laboratory Inspection and Accreditation Program (IAP) has been developed after one year of study supported by a research grant from the Ministry of Health and Welfare (MOHW) of the Republic of Korea from June 1998 to May 1999 to assess objectively the quality of laboratory work and assist the laboratories in improving the quality of their work. The IAP is based on peer review and voluntary participation. The IAP has been continuously improved since the first laboratory inspection began in May 1999 and it was soon expanded nationwide. The improvement was made by updating the inspection checklists to reflect feedback from inspection activities and holding frequent inspectors training workshops. This paper describes the progress and outcome of the IAP. METHODS: The IAP has been implemented nationwide through the following steps: 1) preliminary review of application papers including laboratory quality control policies and external proficiency survey results, as well as on-site inspection by inspectors; 2) addition of newly approved "Inpatient Interpretive Summary Report"checklist (IISR); 3) inspectors training workshop for the "IISR"checklist; 4) continuation of the IAP for all checklist areas including "IISR"; and 5) the first revision of checklists. RESULTS: One hundred nineteen laboratories were accredited during the first year of the IAP. Due to the implementation of the MOHW approved health insurance reimbursement item for laboratory physicians, the "IISR"checklist was created. The mean score of the laboratory inspection results was 92.8 and hospital laboratories showed a higher score on routine testing areas, however, commercial reference laboratories showed a better score on special testing areas. The checklists were revised according to the feedback from the first round of inspections. CONCLUSIONS: The nationwide implementation of the KSLM laboratory IAP was accomplished through this study. The IAP appears to have provided a firm basis for the improvement of quality and efficiency of clinical laboratories in the country.
Accreditation* ; Checklist ; Education ; Financing, Organized ; Insurance, Health, Reimbursement ; Korea ; Laboratories, Hospital ; Peer Review ; Quality Control ; Republic of Korea

BACKGROUND: The Korean Society of Laboratory Medicine (KSLM) Laboratory Inspection and Accreditation Program (IAP) has been developed after one year of study supported by a research grant from the Ministry of Health and Welfare (MOHW) of the Republic of Korea from June 1998 to May 1999 to assess objectively the quality of laboratory work and assist the laboratories in improving the quality of their work. The IAP is based on peer review and voluntary participation. The IAP has been continuously improved since the first laboratory inspection began in May 1999 and it was soon expanded nationwide. The improvement was made by updating the inspection checklists to reflect feedback from inspection activities and holding frequent inspectors training workshops. This paper describes the progress and outcome of the IAP. METHODS: The IAP has been implemented nationwide through the following steps: 1) preliminary review of application papers including laboratory quality control policies and external proficiency survey results, as well as on-site inspection by inspectors; 2) addition of newly approved "Inpatient Interpretive Summary Report"checklist (IISR); 3) inspectors training workshop for the "IISR"checklist; 4) continuation of the IAP for all checklist areas including "IISR"; and 5) the first revision of checklists. RESULTS: One hundred nineteen laboratories were accredited during the first year of the IAP. Due to the implementation of the MOHW approved health insurance reimbursement item for laboratory physicians, the "IISR"checklist was created. The mean score of the laboratory inspection results was 92.8 and hospital laboratories showed a higher score on routine testing areas, however, commercial reference laboratories showed a better score on special testing areas. The checklists were revised according to the feedback from the first round of inspections. CONCLUSIONS: The nationwide implementation of the KSLM laboratory IAP was accomplished through this study. The IAP appears to have provided a firm basis for the improvement of quality and efficiency of clinical laboratories in the country.
Accreditation* ; Checklist ; Education ; Financing, Organized ; Insurance, Health, Reimbursement ; Korea ; Laboratories, Hospital ; Peer Review ; Quality Control ; Republic of Korea

Mycobacterium is an uncommon cause of peritonitis in patients receiving peritoneal dialysis (PD), and the incidence of nontuberculous mycobacterium (NTM) peritonitis is even rarer since the majority of mycobacterial peritonitis cases are caused by Mycobacterium tuberculosis. However, NTM peritonitis has been known to result in a high mortality rate with delayed diagnosis and treatment. In this study, we report a case of Mycobacterium abscessus peritonitis in a 52-year-old male under continuous ambulatory peritoneal dialysis (CAPD).
Delayed Diagnosis ; Humans ; Incidence ; Male ; Mycobacterium ; Mycobacterium tuberculosis ; Nontuberculous Mycobacteria ; Peritoneal Dialysis ; Peritoneal Dialysis, Continuous Ambulatory ; Peritonitis

Listeria grayi is a catalase-positive, non-spore forming, and glucose-fermenting Gram-positive rod. L. grayi is widely distributed in environments such as soil, water and fresh food. Human infection by L. grayi is very rare, and there have been no cases reported in Korea, and only two cases worldwide. Dermabacter hominis is a relatively new species belonging to the coryneform bacteria and is a component of the normal human skin flora. D. hominis is a non-motile, glucose-fermenting, Gram-positive rod that has similar biochemical characteristics to L. grayi. The authors of the present study report a case initially misidentified as L. grayi via a traditional morphological and biochemical identification method but that was subsequently confirmed as D. hominis using sequence analysis of 16S rRNA.
Bacteria ; Humans ; Korea ; Listeria ; Sequence Analysis ; Skin ; Soil

In July 2010, we identified an outbreak of vancomycin-resistant enterococci (VRE) in our 26-bed neonatal intensive care unit. We performed an epidemiological investigation after clinical cultures of 2 neonates were positive for VRE. Identification, susceptibility testing, and molecular characterization were performed. Cultures of 3 surveillance stool samples of inpatients and 5 environmental samples were positive for VRE. All isolates were identified as Enterococcus faecium containing the vanA gene. Two distinct clones were identified by performing pulsed-field gel electrophoresis. The 2 clones exhibited different pulsotypes, but they represented identical Tn1546 types. Two sequence types, ST18 and ST192, were identified among all of the isolates with multilocus sequence typing. Our investigation determined that the outbreak in the neonatal intensive care unit was caused by 2 genetically different clones. The outbreak may have occurred through clonal spread and horizontal transfer of the van gene.
Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/genetics ; Bacterial Typing Techniques ; Carbon-Oxygen Ligases/genetics ; DNA, Bacterial/analysis ; *Disease Outbreaks ; Electrophoresis, Gel, Pulsed-Field ; Enterococcus faecium/drug effects/*genetics/isolation & purification ; Feces/microbiology ; Genotype ; Gram-Positive Bacterial Infections/diagnosis/epidemiology/*microbiology ; Humans ; Infant, Newborn ; Intensive Care Units, Neonatal ; Male ; Multilocus Sequence Typing ; Vancomycin/pharmacology ; *Vancomycin Resistance