Variation and Characterization of Bacterial Communities Contaminating Two Saunas Operated at 64degrees C and 76degrees C

This study was performed to analyze 6 day-term variations in bacterial communities contaminating the floor of two dry saunas that were operated at 64degrees C (low temp) and 76degrees C (high temp). Bacteria were sampled daily from the saunas for 6 days from Monday to Saturday. Genomic DNA was isolated directly from bacteria-collected cotton swabs. The diversity of the bacterial communities collected from the saunas was analyzed using thermal gradient gel electrophoresis (TGGE). The total numbers of DNA bands separated by TGGE for bacteria collected from the low temp and high temp sauna were 20 and 18, respectively, during the 6 days. Seven of 20 bacteria in the low temp sauna and eight of 18 bacteria in the high temp sauna were detected more than three times over the 6 experimental days. Twelve of the 26 bacterial genera contaminating the saunas were cross detected. Bacteria belonging to the genera Moraxella and Acinetobacter were selectively detected in the low temp sauna, whereas those belonging to Aquaspirillum, Chromobacterium, Aquabacterium, Gulbenkiania, Pelomonas, and Aquitalea were selectively detected in the high temp sauna. Three species of bacteria contaminating both the low and high temp saunas were thermophile or thermoduric. The results indicate that the sauna-contaminating bacteria may have been transferred from outside the saunas by user traffic but did not inhabit the saunas.

Detection of Botulinum Neurotoxin Type A by In Vitro Bioassay Based on Endopeptidase Activity

Botulinum neurotoxin type A (BoNT/A) is a metalloprotease that cleaves SNAP-25 (synaptosome-associated protein of 25 kDa), a specific cellular protein essential for neurotransmitter release. As well as mouse bioassay to detect BoNT/A, various assay methods based on its endopeptidase activity have been developed. In this study, we tried to develop a BoNT/A assay system using recombinant SNAP-25 with glutathione S-transferase (GST) tags at both termini as substrate. The recombinant GST-SNAP-25-GST with 70 kDa was expressed and purified in E. coli and synthesized N-terminal 50 kDa and C-terminal 25 kDa fragment after cleavage at the Gln(197)-Arg(198) bond by BoNT/A. To detect both fragments, we obtained rabbit antisera against peptides corresponding to the cleaved ends of each fragment. In the western blotting, the N-terminal fragment was detected by the antibody specifically recognizing the newly exposed C-terminus (corresponding to amino acid residue 191-197). This assay system was able to detect until 3.125 ng of BoNT/A, which corresponded to about 90 fold LD50 in mice. These results suggest that the in vitro endopeptidase assay developed in this study would replace others to detect BoNT/A.

Detection of Botulinum Neurotoxin Type A by In Vitro Bioassay Based on Endopeptidase Activity

Botulinum neurotoxin type A (BoNT/A) is a metalloprotease that cleaves SNAP-25 (synaptosome-associated protein of 25 kDa), a specific cellular protein essential for neurotransmitter release. As well as mouse bioassay to detect BoNT/A, various assay methods based on its endopeptidase activity have been developed. In this study, we tried to develop a BoNT/A assay system using recombinant SNAP-25 with glutathione S-transferase (GST) tags at both termini as substrate. The recombinant GST-SNAP-25-GST with 70 kDa was expressed and purified in E. coli and synthesized N-terminal 50 kDa and C-terminal 25 kDa fragment after cleavage at the Gln(197)-Arg(198) bond by BoNT/A. To detect both fragments, we obtained rabbit antisera against peptides corresponding to the cleaved ends of each fragment. In the western blotting, the N-terminal fragment was detected by the antibody specifically recognizing the newly exposed C-terminus (corresponding to amino acid residue 191-197). This assay system was able to detect until 3.125 ng of BoNT/A, which corresponded to about 90 fold LD50 in mice. These results suggest that the in vitro endopeptidase assay developed in this study would replace others to detect BoNT/A.

Development of Capture ELISA Using a Biotinylated Monoclonal Antibody for Detection of Botulinum Neurotoxin Type A

A capture enzyme-linked immunosorbent assay (capture ELISA) was developed to detect Clostridium botulinum neurotoxin type A (BoNT/A) in assay buffer and human serum. The assay is based upon affinity-purified rabbit polyclonal and biotinylated monoclonal antibodies directed against the BoNT/A complex purified from C. botulinum ATCC19397. For the capture ELISA, the optimized amount (2 microgram/ml) of rabbit polyclonal antibody was immobilized on ELISA plates to detect BoNT/A (ranging from 0 to 500 ng/ml), which was recognized by 2 microgram/ml of the monoclonal antibody. From three independent repeated experiments, standard curves were linear over the range of 0~31.25 ng/ml BoNT/A and the coefficients (r(2)) ranged from 0.9951~0.9999 for all assays. The inter-variations were typically 0.50~6.93% and the specificity was confirmed by showing no cross-reactivity against BoNT/B and /E. The detection limit of capture ELISA was 0.488 ng/ml, which was close to mouse LD(50). In addition, application with BoNT/A-spiking human sera showed a possibility to detect BoNT/A with capture ELISA from the contaminated human sera. Taken together, the newly developed capture ELISA could serve as a rapid and sensitive screening tool for detecting BoNT/A simultaneously from massive specimens.

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

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 / 감염과화학요법

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.

The Present Situation of Infection Control Professionals, Organization, and Activities in Korean Acute Care General Hospitals / 병원감염관리

BACKGROUND: It has been more than 15 years since infection control was first introduced in Korea, but there is little information available on the status of infection control program in the country. METHODS: Included in the study were 139 acute care hospitals with more than 300 inpatient beds. A questionnaire, modified from US SENIC (Study on the Efficacy of Nosocomial Infection Control) and Canadian RICH (Resources for Infection Control in Canadian Acute Care Hospitals) survey, was mailed to the hospitals in the winter of 2003. RESULTS: Ninety-eight (70.5%) of 139 hospitals responded. There was an average of 1.2 (SD, 0.7) Infection Control Practitioners (lCPs) in each hospital and 95.7% were nurses and only 56.5% of the ICPs worked as full-time. The 71.4% of the hospitals had a position for Infection Control Doctor. All hospitals had an Infection Control Committee, which met an average of 3.7 (SD, 1.7) times a year. The 85.7% of the hospitals performed surveillance, but only 31.6% were monitoring surgical site infections. Review of microbiology data was the most common method for case-finding. More than 90% of the hospitals had infection control policies and guidelines, but an adherence to the policies and guidelines was not monitored regularly. CONCLUSION: This study reports the first comparable profile of infection control program of general acute care hospitals in Korea. Although the foundation for infection control program appears to have been established, there is the need for a further increase in the number of ICPs, the standardization of the surveillance method, and the promotion of adherence to the infection control guidelines.

Prevalence and Genetic Relatedness of Vancomycin-resistant Enterococci Isolated from Livestock and Humans after Ban of Avoparcin in Korea / 감염과화학요법

BACKGROUND: Avoparcin, cross-resistance with vancomycin, was added as feed-additive since 1970s and was prohibited in 1997 in Korea. After avoparcin was banned we examined prevalence and genetic relatedness of VRE in enterococci isolated from livestock and humans. MATERIALS AND METHODS: Using enrichment broth and 6 microgram/mL vancomycin-containing enterococcosel selective agar, vancomycin-resistant enterococci (VRE) were isolated from fecal sample of 255 pigs of 8 farms, 431 chickens of 9 farms, and 328 humans (Food industry employee and Institution cafeteria employee) of 5 public health centers, and 100 raw chicken meats from April to June 2003. Antimicrobial susceptibility was examined by disk diffusion and minimum inhibitory concentrations (MICs), and E-test. Species identification and genotyping were done by multiplex PCR method. Pulsed-field gel electrophoresis (PFGE) of vanA-type VRE isolates was performed by CHEF-Mapper system. RESULTS: 19 isolates from 255 pigs, 122 isolates from 431 chickens, 19 isolates from 100 raw chicken meat, and 7 isolates from 328 humans were resistant to vancomycin. Of the 167 VRE isolates, vanA gene was detected in 141 isolates; 1 isolate (0.4%) in pigs, 121 isolates (28.1%) in chickens, 18 isolates (18.0%) in raw chicken meat, and 1 isolate (0.3%) in humans. Resistant rates of streptomycin, tetracycline, and erythromycin were over 60% in vanA-type E. faecium isolated from poultry. PFGE analysis resulted in two major patterns, F and P types. Also PFGE pattern of 1 VRE from human was identical to that of 1 VRE from poultry. CONCLUSION: Despite the high prevalence of vanA-type VRE in poultry farms, VRE isolation rate in human was relatively low. This result suggests that the possibility of VRE transmission from poultry to human is low but that possibility may be not ruled out. In PFGE analysis showing 51.5% identical in 2 PFGE patterns, the dissemination of VRE isolates in poultry may be transmitted vertically and horizontally.

Antimicrobial Resistance among Clinical Isolates of Pseudomonas aeruginosa from Non-tertiary Care Hospitals in Korea, 2002-2004 / 감염과화학요법

BACKGROUND: Increasing numbers of resistant and multidrug resistant (MDR) isolates of Pseudomonas aeruginosa have become a worldwide problem. This report provides the trend of antimicrobial resistance, the proportions of MDR and metallo-beta-lactamase-producing isolates among clinical isolates of P. aeruginosa in Korea. MATERIALS AND METHODS: Clinical isolates of P. aeruginosa were collected from two representative reference laboratories during 2002-2004. Clinical information regarding specimens and type of hospital for isolates was investigated. Antimicrobial susceptibility against 11 antibiotics was tested by disk diffusion according to NCCLS criteria. MDR was assessed as resistance to > or =3 of the core drugs (ceftazidime, ciprofloxacin, gentamicin, imipenem and piperacillin). PCR assays and sequencing for detection of blaVIM-2 and blaIMP-1 gene were carried out. RESULTS: Of 1,748 P. aeruginosa isolates, 179 isolates were collected from primary care hospitals and 1,569 isolates were recovered from outpatients and inpatients in secondary care hospitals. From 2002 to 2004, rates of resistance to ceftazidime and imipenem increased from 10% to 12.3% and from 14.8% to 15.9%, respectively. Rates of resistance to amikacin (from 26.2% to 31.0%) and ciprofloxacin (from 35.6% to 46.2%) increased annually. In the period 2002-2004, decreasing of susceptibility to meropenem (from 83.4% to 76.8%) was observed, but meropenem was the most potent agent against P. aeruginosa isolates studied. During the 3-year period, MDR P. aeruginosa accounted for 26.4-33.5% of clinical isolates and the most common MDR phenotype was concurrent resistance to piperacillin, gentamicin and ciprofloxacin. The prevalence of VIM-2-producing isolates obviously increased from 1.7% in 2002 to 6.3% in 2004. CONCLUSIONS: These results suggested that MDR P. aeruginosa was already prevalent in one third of clinical isolates and VIM-2-producing P. aeruginosa isolates disseminated in non-tertiary care hospitals in Korea.

Prevalence and Genetic Relatedness of Vancomycin-resistant Enterococci Isolated from Livestock and Humans after Ban of Avoparcin in Korea / 감염과화학요법

BACKGROUND: Avoparcin, cross-resistance with vancomycin, was added as feed-additive since 1970s and was prohibited in 1997 in Korea. After avoparcin was banned we examined prevalence and genetic relatedness of VRE in enterococci isolated from livestock and humans. MATERIALS AND METHODS: Using enrichment broth and 6 microgram/mL vancomycin-containing enterococcosel selective agar, vancomycin-resistant enterococci (VRE) were isolated from fecal sample of 255 pigs of 8 farms, 431 chickens of 9 farms, and 328 humans (Food industry employee and Institution cafeteria employee) of 5 public health centers, and 100 raw chicken meats from April to June 2003. Antimicrobial susceptibility was examined by disk diffusion and minimum inhibitory concentrations (MICs), and E-test. Species identification and genotyping were done by multiplex PCR method. Pulsed-field gel electrophoresis (PFGE) of vanA-type VRE isolates was performed by CHEF-Mapper system. RESULTS: 19 isolates from 255 pigs, 122 isolates from 431 chickens, 19 isolates from 100 raw chicken meat, and 7 isolates from 328 humans were resistant to vancomycin. Of the 167 VRE isolates, vanA gene was detected in 141 isolates; 1 isolate (0.4%) in pigs, 121 isolates (28.1%) in chickens, 18 isolates (18.0%) in raw chicken meat, and 1 isolate (0.3%) in humans. Resistant rates of streptomycin, tetracycline, and erythromycin were over 60% in vanA-type E. faecium isolated from poultry. PFGE analysis resulted in two major patterns, F and P types. Also PFGE pattern of 1 VRE from human was identical to that of 1 VRE from poultry. CONCLUSION: Despite the high prevalence of vanA-type VRE in poultry farms, VRE isolation rate in human was relatively low. This result suggests that the possibility of VRE transmission from poultry to human is low but that possibility may be not ruled out. In PFGE analysis showing 51.5% identical in 2 PFGE patterns, the dissemination of VRE isolates in poultry may be transmitted vertically and horizontally.

Antimicrobial Resistance among Clinical Isolates of Pseudomonas aeruginosa from Non-tertiary Care Hospitals in Korea, 2002-2004 / 감염과화학요법

BACKGROUND: Increasing numbers of resistant and multidrug resistant (MDR) isolates of Pseudomonas aeruginosa have become a worldwide problem. This report provides the trend of antimicrobial resistance, the proportions of MDR and metallo-beta-lactamase-producing isolates among clinical isolates of P. aeruginosa in Korea. MATERIALS AND METHODS: Clinical isolates of P. aeruginosa were collected from two representative reference laboratories during 2002-2004. Clinical information regarding specimens and type of hospital for isolates was investigated. Antimicrobial susceptibility against 11 antibiotics was tested by disk diffusion according to NCCLS criteria. MDR was assessed as resistance to > or =3 of the core drugs (ceftazidime, ciprofloxacin, gentamicin, imipenem and piperacillin). PCR assays and sequencing for detection of blaVIM-2 and blaIMP-1 gene were carried out. RESULTS: Of 1,748 P. aeruginosa isolates, 179 isolates were collected from primary care hospitals and 1,569 isolates were recovered from outpatients and inpatients in secondary care hospitals. From 2002 to 2004, rates of resistance to ceftazidime and imipenem increased from 10% to 12.3% and from 14.8% to 15.9%, respectively. Rates of resistance to amikacin (from 26.2% to 31.0%) and ciprofloxacin (from 35.6% to 46.2%) increased annually. In the period 2002-2004, decreasing of susceptibility to meropenem (from 83.4% to 76.8%) was observed, but meropenem was the most potent agent against P. aeruginosa isolates studied. During the 3-year period, MDR P. aeruginosa accounted for 26.4-33.5% of clinical isolates and the most common MDR phenotype was concurrent resistance to piperacillin, gentamicin and ciprofloxacin. The prevalence of VIM-2-producing isolates obviously increased from 1.7% in 2002 to 6.3% in 2004. CONCLUSIONS: These results suggested that MDR P. aeruginosa was already prevalent in one third of clinical isolates and VIM-2-producing P. aeruginosa isolates disseminated in non-tertiary care hospitals in Korea.

Antimicrobial Resistance of Clinical Isolates of Acinetobacter spp. Collected from Non-Tertiary Hospitals and Detection of a Metallo-beta-Lactamase-Producing Strain / 감염과화학요법

BACKGROUND: The aim of this study was to investigate the antimicrobial resistance of clinical isolates of Acinetobacter spp. collected from non-tertiary hospitals and to characterize the phenotype and the genotype of imipenem-non-susceptible isolates. MATERIALS AND METHODS: Clinical isolates of Acinetobacter spp. were identified using recA-restriction fragment length polymorphism (RFLP) analysis with Tsp5091. Susceptibility to antimicrobial agents was determined using disk diffusion test and agar dilution test according to the criteria of the National Committee for Clinical Laboratory Standards. PCR and sequence analyses were used to detect the blaIMP-1 and blaVIM-2 genes, and to determine the content and order of the resistance genes inserted in integron. RESULTS: Of 71 Acinetobacter spp. isolates collected from non-tertiary hospitals during 2002 and 2003, 60 isolates were A. baumannii, and 2, 4, and 5 isolates were Acinetobacter genomic species 3, 13TU, and A. lwoffii, respectively. The resistance rate of Acinetobacter spp. isolates to beta-lactams, aminoglycosides, and fluoroquinolones was high except for imipenem and meropenem. The presence of blaVIM-2 gene was found in one isolate, Acinetobacter genomic species 13TU, for which the MIC of imipenem was 8 mg/L; the blaVIM-2 gene of this strain was located on 3 kb class 1 integron with aacA7 and aadA1 genes. CONCLUSIONS: Among the tested agents, imipenem and meropenem retained greatest activity against Acinetobacter spp. isolates collected from non-tertiary hospitals. This is the first report of VIM-2-producing Acinetobacter genomic species 13TU strains with class 1 integron containing blaVIM-2 gene.

Antimicrobial Resistance of Clinical Isolates of Acinetobacter spp. Collected from Non-Tertiary Hospitals and Detection of a Metallo-beta-Lactamase-Producing Strain / 감염과화학요법

BACKGROUND: The aim of this study was to investigate the antimicrobial resistance of clinical isolates of Acinetobacter spp. collected from non-tertiary hospitals and to characterize the phenotype and the genotype of imipenem-non-susceptible isolates. MATERIALS AND METHODS: Clinical isolates of Acinetobacter spp. were identified using recA-restriction fragment length polymorphism (RFLP) analysis with Tsp5091. Susceptibility to antimicrobial agents was determined using disk diffusion test and agar dilution test according to the criteria of the National Committee for Clinical Laboratory Standards. PCR and sequence analyses were used to detect the blaIMP-1 and blaVIM-2 genes, and to determine the content and order of the resistance genes inserted in integron. RESULTS: Of 71 Acinetobacter spp. isolates collected from non-tertiary hospitals during 2002 and 2003, 60 isolates were A. baumannii, and 2, 4, and 5 isolates were Acinetobacter genomic species 3, 13TU, and A. lwoffii, respectively. The resistance rate of Acinetobacter spp. isolates to beta-lactams, aminoglycosides, and fluoroquinolones was high except for imipenem and meropenem. The presence of blaVIM-2 gene was found in one isolate, Acinetobacter genomic species 13TU, for which the MIC of imipenem was 8 mg/L; the blaVIM-2 gene of this strain was located on 3 kb class 1 integron with aacA7 and aadA1 genes. CONCLUSIONS: Among the tested agents, imipenem and meropenem retained greatest activity against Acinetobacter spp. isolates collected from non-tertiary hospitals. This is the first report of VIM-2-producing Acinetobacter genomic species 13TU strains with class 1 integron containing blaVIM-2 gene.

Frequency of Resistance to Aminog lycoside Antibiotics in Staphy lococcus aureus Isolated from Tertiary Hospitals / 감염

BACKGROUND: Staphylococcus aureus is one of the most important pathogens, causing severe morbidity and fatal infections. To date rapid evolution of antibiotic resistance in S. aureus, including recent emergence of vancomycin-resistant S. aureus (VRSA), has been a serious concern and an obstacle to the effective treatment. The purpose of this study is to update the resistance patterns against aminoglycoside antibiotics which play an important role in the therapy of serious staphylococcal infections. METHODS: Clinical isolates were collected from 8 university-affiliated hospitals during the period of June 1999 to January 2001. Susceptibility tests against 9 antibiotics were performed by disk diffusion method. Minimum inhibitory concentrations (MICs) of arbekacin against non-susceptible strains were determined by microbroth dilution method RESULTS: Among total 682 isolates exclusive of consecutive ones from the same patients, 199 (29%) were from pus, 152 (22%) from respiratory specimens, 137 (20%) from blood, 38 (6%) from urine. Of 682 isolates, 588 (87%) isolates were resistant to at least one of the aminoglycosides tested. Overall prevalence of MRSA was 64% (439/682), and resistance rates of MRSA were summarized as follows; kanamycin (KM) 98%, tobramycin (TOB) 98%, gentamicin (GM) 95%, amikacin (AMK) 90%, neomycin (NEO) 63%, streptomycin (SM) 31%, netilmicin (NET) 18%, arbekacin (ABK) 13%. MRSA isolates were resistant to multiple aminoglycosides, and 88% of them were resistant to all four aminoglycosides of KM, TOB, GM, and AMK. MICs of ABK against 58 non-susceptible strains ranged from 2 to 128 microgram/mL. CONCLUSION: More than 90% of MRSA isolates were resistant against kanamycin, tobramycin, gentamicin, and amikacin. Moreover, most of MRSA isolates were multi-drug resistant to all these four aminoglycosides. Resistance rates against arbekacin and netilmicin were less than 20%. Arbekacin was the most susceptible antibiotic of the aminoglycosides tested.

Usefulness of PCR Method for Identification of Enterococci Species / 감염

BACKGROUND: Enterococci are important cause of nosocomial infections. Recently, vancomycin-resistant enterococci (VRE) has been increasingly reported as significant nosocomial pathogens. Therefore, accurate identification of enterococcal species is a prerequisite step for the appropriate antibiotic treatment and epidemiologic surveillance. We wanted to know the usefulness of PCR method compared with Vitek automatic identification system. METHODS: Totally 105 isolates were identified on the species level by Vitek (GPI card and software version R06.1), methyl-alpha-D-glucopyranoside test, and PCR methods. RESULTS: Among 105 enterococcal isolates, 59 were identified as E. faecium, 11 E. faecalis, 6 E. gallinarum by Vitek. But 29 isolates (28%) were unidentified. Subsequently all of these isolates were analyzed by PCR, the results of which were as follows: 17 E. faecium, 5 E. casseliflavus, 7 E. gallinarum. Two isolates identified as E. gallinarum by Vitek were reidentified as E. casseliflavus by PCR and other methods for phenotypic characterization. CONCLUSOIN: PCR method was more accurate and sensitive than Vitek for the identification of enterococci species.

Molecular Relationship of vanA Glycopeptide Resistance Gene in Enterococci from Hospitalized Patients and Poultry in Korea / 감염

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.

Antibiotic Resistance Patterns of Staphylococcus aureus Isolated from the Patients Admitted to Non-tertiary Hospitals / 감염

BACKGROUND: About more than 70% of Staphylococcus aureus isolates in tertiary-care hospitals are known to be resistant to methicillin in Korea. But the prevalence of methicillin-resistant S. aureus (MRSA) in the community and non-tertiary hospitals has not been known yet. The aim of this study was to determine the prevalence of resistance among S. aureus isolates in non-tertiary hospitals. METHODS: The isolates were collected at one laboratory center from August 1998 to May 1999. Antimicrobial susceptibility tests against 11 antibiotics were performed by disk diffusion method. Minimum inhibitory concentrations (MIC) for oxacillin and vancomycin were determined by microbroth dilution method. The mecA gene was detected by polymerase chain reaction. The medical facilities which sent specimen to the laboratory were classified into 3 groups; clinic, hospital and general hospital. RESULTS: Of total 469 S. aureus isolates, 296 (63.1%) were from pus, 47 (10.0%) from sputum, 23 (5.0%) from urine, and 22 (4.6%) from blood. Overall prevalence of MRSA in non-tertiary hospital was 43.5% (204/469). Among 3 hospital groups, MRSA in general hospitals (55%) was significantly more prevalent than in hospitals (40%) or clinics (37%). MICs of oxacillin against MRSA isolated from pus and blood ranged from 8 to > or =256 microgram/mL, but 74% (83 isolates) of them was > or =256 microgram/mL. MICs of vancomycin were distributed from 1 to 2 microgram/mL, irrespective of methicillin resistance or hospital groups. The mecA gene was detected in all of methicillin-resistant isolates with MICs of < or =128 microgram/mL. CONCLUSION: In non-tertiary hospitals, 43% of S. aureus isolates were methicillin resistant. This result showed that MRSA in non-tertary hospitals was less prevalent than in tertiary hospitals.

Nosocomial Infection Research Activities in Laboratory of Nosocomial Infection Pathogens in NIH of Korea / 병원감염관리

No Abstract available.

Purification of enolase from Candida albicans KNIH10 isolated in Korea and application of immunological diagnosis

We purified enolase from Candida albicans KNIH10 strain which was isolated from a clinical specimen in Korea. The purified enolase was used to detect anti-Candida antibodies in sera of patients with invasive candidiasis. For purification of enolase from the crude extract prepared by French pressure at 20,000 PSI, the fast performance liquid chromatography (FPLC) using DEAE-sepharose column was used. The elutes at 0.3-0.4 M NaCl in FPLC was purified with homogenity in SDS-PAGE and its enzymatic activity was confirmed in sera of invasive candidiasis with candidemia patient by immunoblotting. The purified enolase indicated no siggnal (100% specificity) in 40 normal human sera and 75% (6/8) sensitivity in sera of candidemic patients with suspicious invasive candidiasis by immunoblotting.

Identification and phylogenetic relationship of dermatophytes based on RFLP analysis and nucleotide sequence of internal transcribed spacer (ITS)1 in nuclear ribosome DNA

ITSI-5.8S-ITSII rDNA region was amplified from the reference strains and clinical isolates with ITS1 and ITS4 primers. These primers amplified DNA fragments of 550 bp in Microsporum audouinii and Trichophyton violaceum, 700 bp in Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum, and Trichophyton tonsurans, and 750 bp in Microsporum ferreugineum and Microsporum canis. The restriction enzyme patterns of PCR products digested with 13 restriction enzyme including PstI were distint among the genera, whereas identical in the same species. Examination of the ITS (Internal Transcribed Spacers)1 nucleotide sequence revealed that there was the genetic difference in each genera and species. Phylogenetic relationship among each species showed that the Trichophyton mentagrophytes was more closely related Trichophyton tonsurans than Trichophyton rubrum, and Microsporum gypseum was less related than Microsporum spp.