국내 분리 렙토스피라균의 단클론 항체 및 Genomic DNA의 Pulsed-Field Gel Electrophoresis 분석

조민기,기선호,김형준,김윤원,장우현,오희복 한국미생물학회 1999 미생물학회지 Vol.35 No.3

1996년 경기도, 강원도 충청북도, 전라남도, 전라북도 등 일부지역에서 채집된 들쥐들로부터 분리된 22주의 렙토스피라균의 단클론 항체에 대한 반응양상 및 genomic DNA 의 pulsed-field gel electrophoresis pattern을 분석하였다. 혈청군 Icterohemorrhagiae 내의 균주로 면역하여 제조한 7가지 단클론 항체에 대한 분리균주들의 반응은 모두 혈청형 lai 와 같은 pattern을 보였다. Not I 제한효소 절단 DNA 의 PFGE에서 JR34, JR57, JR77, JT82, JR86, JR109, NR4, NR6, NR13, CR3, KR48, NR2, NR8, NR9, NR10, NR11, NR12, JR58, 및 JR62 주 들은 모두 혈청형 lai 와 유사한 profile을 보였으며 940 kb와 63kb 사이에 13개의 절편 band를 보였다. JR89주는 혈청형 lai 및 다른 분리균주에서 관찰되지 않은 1000 kb band 와 460 kb band 가 관찰되었다.표준균주 혈청형 lai, birkini, gem, mwogolo, canicola 등은 각기 완전히 다른 pattern을 보였으며 혈청형 yeonchon 은 lai 와 같은 pattern을 보였다. UPGMA방법에 의한 dendrogram 분석결과 분리주는 lai 및 yeonchon 혈청형과 81~85%, 기타 혈청형과는 62% 이하의 유사도를 나타내어 항원분석법에 의한 혈청형 동정결과와 일치하였다. Asc I 제한요소 절단 DNA 의 PFGE에서는 JR34, JR77, JR82, JR109, NR6, NR13, CR3, KR48, 및 JR57, JR58, JR62, JR86, NR2, NR3, NR4, NR8, NR9, NR10, NR11, NR12 주들은 모두 1900 kb 에서부터 380kb 사이에 3개의 절편 band를 보였으며 이는 표준균주 lai 와 같은 pattern 이었다. JR89주는 다른 분리균주와는 달리 1640 kb 대신 650kb 의 band를 보였다. Fse I 제한효소 절단 PFGE 에서는 JR57, JR77, JR82, JR86, JR109, NR4, NR6, NR13, CR3, KR48주 및 JR34, NR2, NR3, NR9, NR10 주들은 모두 1900 kb 와 280kb 사이에 5개의 절편 band를 보였으며 이는 표준균주 lai 및 yeonchon 과 같은 pattern 이었다. 그러나 JR89주에서는 280kb 가 나타나지 않아 다른 분리균주와 구분되었다. A total of 22 Leptospiua inlermgans field isolates from the ~ a t s captured in 5 provinces of Korea in 1996, and 6 antigenically closely related relerence serovars of lai, yeonchon, birkini. gem, mwogolo. and canicola were analysed. When the antigenic characteristics were analysed by reactivity with 7 monoclonal antibodies prepared with sh.ains belongng to serogroup Icterohaemorrhagiae. all 22 isolates showed the same reaction pattern with that of serovar lai. Large restriction fragment patterns obtained after cleavage of geno~nic DNAs with infrequently cuttimg restriction enzymes were analyzed by pulsed-field pel electrophoresis(PFGE). Identification of leptospira strains by PFGE with Nor I, Asc I or Iise I digests correlated with their antigenically typed serovars, silh a few exceptions. PFGE of isolates, except for JR89, digested wjth Nor I showed identical pattern w~th serovar lai, showing 13 Cragments between 940 kb and 63 kb. When PFGE pallerns of JR89 were compared with those of serovar lai, Not I digest showed additional two hands of 1000 kb and 460 kb, while Asc I digest showed 650 kb fragment and Fse I digest did not show the fragment of 280 kb. Whereas serovar yeonchon. which was isolated in Korea and identified as a new serovar previously. could be differentiated from serovar lai in antigenic reactivities with monoclonal antibodres. it showed the similar PFGE pattern with serovar lai includin~ reference and field isolates. It was suggested that Korean leptospiral field isolates are closely related in DNA level.

Pulsed-field gel electrophoresis를 이용한 중환자실 Serratia marcescens 집단요로감염의 역학적 조사

고은하,김선주,배인규 대한임상미생물학회 2005 Annals of clinical microbiology Vol.8 No.1

Background: Serratia marcescens is a well-known cause of nosocomial infections. We investigated an outbreak of S. marcescensinfections in a surgical intensive care unit (SICU) and identified the source of the outbreak using pulsed-field gel electrophoresis (PFGE). Methods: A total of 39 isolates of S. marcescenswere included in this study: 28 isolates from the patients in the SICU and epidemiologically-unrelated 11 isolates from the patients in the general wards from May through August, 2003 at Gyeongsang National University Hospital. Twenty-six of the 28 isolates in the SICU were from the urine collected from indwelling urinary catheters. Fifty-six environmental samples, such as the hands of healthcare workers and urinals were cultured to identify the source of infection. Antimicrobial susceptibility tests by Vitek GNS card (bioMerieux) and PFGE were performed to identify the clonality of the isolates. Results: Twenty of the 28 S. marcescens isolated from the patients in the SICU showed the identical PFGE fingerprint pattern and two isolates had a closely-related pattern with the outbreak strain. The isolates from urine in the SICU were resistant to almost all the antibiotics tested except imipenem and cotrimoxazole. Nine of the 11 isolates from the general wards had PFGE patterns and antimicrobial susceptibility results different from those of the outbreak clone. Five samples from used-urinals and one from disinfected-urinal of 56 environmental samples grew S. marcescensthat were resistant to the all antibiotics tested except imipenem and cotrimoxazole. Conclusion: The outbreak of urinary tract infections in SICU was due to a clonal spread of a single strain of S. marcescens that was multiple resistant to antibiotics except imipenem and cotrimoxazole. The source of outbreak appeared to be inadequately disinfected urinals.

서울시에서 산발적으로 분리된 Salmonella enterica Serotype Typhi 임상분리균주들의 분자역학적 분석

이희무,김철현,손장욱,김민자,박미선,이복권 대한감염학회 2000 감염 Vol.32 No.5

Comparison of Modified Multiple-locus Variable-number Tandem-repeat Fingerprinting with Pulsed-field Gel Electrophoresis for Typing Clinical Isolates of Staphylococcus aureus

정소이,이종윤,장미희,주세익,라은경,김소연,장철훈,박성섭,김의종 대한진단검사의학회 2012 Annals of Laboratory Medicine Vol.32 No.1

Background: Multiple-locus variable-number tandem-repeat fingerprinting (MLVF) is based on multiplex PCR, utilizing variable number tandem repeat. Our goal was to compare the performance of MLVF in distinguishing clinical Staphylococcus aureus isolates with that of pulsed-field gel electrophoresis (PFGE), which has traditionally been the gold standard. Methods: Sixty-three clinically significant S. aureus isolates were tested using both PFGE and MLVF. Multiplex PCR for MLVF was performed using PCR primers for clfA, clfB, sdrCDE, sspA, and spa. PFGE was performed with genomic DNA fragments generated by SmaI endonuclease digestion. Banding patterns of MLVF or PFGE were analyzed using InfoQuestFP software. Results: The hands-on time of our modified method was about 3 h, on average, for each of 18 isolates. PFGE (80% cutoff) or MLVF (75% cutoff) separated all of the 63 isolates into 13 and 12 types, respectively. Three types generated by PFGE were identical to those generated by MLVF. PFGE and MLVF yielded similar Simpson’s diversity indices, indicating similar discriminatory power. The overall concordance between PFGE and MLVF was low, as represented by adjusted Rand indices (0.266-0.278). PFGE predicted MLVF type better than MLVF predicted PFGE type, as reflected by Wallace coefficients (PFGE cutoff 80% vs. MLVF cutoff 75%, 0.389 vs. 0.233). Analysis of the relationship between a pair of isolates showed 91.0% concordance between the PFGE (80% cutoff) and MLVF (75% cutoff). Conclusions: Our simple, low-cost, modified MLVF protocol can effectively discriminate between S. aureus clinical isolates. MLVF can replace PFGE for the hospital infection control of S. aureus. Background: Multiple-locus variable-number tandem-repeat fingerprinting (MLVF) is based on multiplex PCR, utilizing variable number tandem repeat. Our goal was to compare the performance of MLVF in distinguishing clinical Staphylococcus aureus isolates with that of pulsed-field gel electrophoresis (PFGE), which has traditionally been the gold standard. Methods: Sixty-three clinically significant S. aureus isolates were tested using both PFGE and MLVF. Multiplex PCR for MLVF was performed using PCR primers for clfA, clfB, sdrCDE, sspA, and spa. PFGE was performed with genomic DNA fragments generated by SmaI endonuclease digestion. Banding patterns of MLVF or PFGE were analyzed using InfoQuestFP software. Results: The hands-on time of our modified method was about 3 h, on average, for each of 18 isolates. PFGE (80% cutoff) or MLVF (75% cutoff) separated all of the 63 isolates into 13 and 12 types, respectively. Three types generated by PFGE were identical to those generated by MLVF. PFGE and MLVF yielded similar Simpson’s diversity indices, indicating similar discriminatory power. The overall concordance between PFGE and MLVF was low, as represented by adjusted Rand indices (0.266-0.278). PFGE predicted MLVF type better than MLVF predicted PFGE type, as reflected by Wallace coefficients (PFGE cutoff 80% vs. MLVF cutoff 75%, 0.389 vs. 0.233). Analysis of the relationship between a pair of isolates showed 91.0% concordance between the PFGE (80% cutoff) and MLVF (75% cutoff). Conclusions: Our simple, low-cost, modified MLVF protocol can effectively discriminate between S. aureus clinical isolates. MLVF can replace PFGE for the hospital infection control of S. aureus.

신생아 중환자실에서 extended spectrum β-lactamase를 생성하는 Klebsiella pneumoniae 집단 보균 발생의 분자 역학적 조사 및 추적관찰

전누리,김미나,정재심,김양수,김애란,김기수,피수영,Jun, Nu-Lee,Kim, Mi-Na,Jeong, Jae-Sim,Kim, Yang-Soo,Kim, Ellen Ai-Rhan,Kim, Ki-Soo,Pi, Soo-Young 대한소아청소년과학회 2006 Clinical and Experimental Pediatrics (CEP) Vol.49 No.2

목 적 : 2000년 6월과 7월에 본원 신생아 중환자실에서는 ESBL 생성 K. pneumoniae에 의한 패혈증이 집단 발생하여 전체 환아에 대한 보균 상태를 파악하고 감염관리를 위해 감시배양을 실시하였다. 당시 집단 보균 상태임을 발견하여 보균자에 대한 격리를 실시한 결과 집단보균을 해결할 수 있었으나 대부분의 환아가 보균 상태로 퇴원하였다. ESBL 생성 K. pneumoniae는 대변 내 균이 장착되어서 입원 환아들간에 집단 감염을 일으킬 수 있으며, 이러한 균주에 의한 집단감염은 치료와 예후에 중요한 인자로 작용될 것으로 생각된다. 이에 저자들은 집단보균 발생시 분리된 ESBL 생성 K. pneumoniae의 분자 역학적 특징과 보균환아들의 추적관찰 결과를 알아보고자 본 연구를 시행하였다. 방 법 : 2000년 7월 28일부터 12월 30일까지 입원환아를 대상으로 직장내 도말법으로 감시배양검사를 시행하였다. 감시배양검사는 재원환아들에서 1주 간격으로 시행하였고, 신환은 입원 당일에 시행하였다. 균주의 형별 검사를 위해서 Pulsed-field gel electrophoresis(PFGE)를 시행하였고, 보균 상태로 퇴원했던 환아들은 외래에서 대변 배양검사로 추적 관찰하였다. 결 과 : 감시 배양기간 중 총 80명(28.5%)의 환아에서 보균이 확인되었고, 퇴원시 5명(6.3%)에서 음성이 확인되었다. PFGE를 시행한 65명의 타이핑 결과, 일회의 PFGE를 시행한 53명에서 분리된 균주의 염색체 유전자 분획양상은 집단클론 6가지, 단독클론 10가지형으로 분류되었고, 집단 클론 중 A형이 28명(52.8%)으로 가장 많았고, B형이 11명(20.8%), C, D, F, G형이 각각 1명(1.9%)이었다. 2회 이상 검사를 시행한 12명 중 초기검사에서는 A형이 10명(83.3%)으로 월등히 많았고 B형은 2명(16.7%)이었으며, 추적검사에서 분획 양상이 변화된 경우는 6명(50%)이었고, 이들은 A나 B로 변화된 경우가 각각 2명(33.3%)이었다. 변화되지 않은 6명(50%)은 모두 A형으로 남아 있었다. 월별 PFGE 양상은 처음 배양시 집단클론 A, B, C, D형 4가지와 단독클론 세 가지형이었으나 감염 관리를 하면서 11월부터 집단클론 A, B 두 가지형으로 나타나는 양상을 보였고, A형이 더 우세하였다. 보균된 상태로 퇴원한 75명 중 외래 추적관찰이 가능했던 30명을 대상으로 대변 배양검사를 시행한 결과 모두 음성이 확인되었다. 결 론 : 다양한 클론의 균주에 의한 집단보균 상태는 감염 관리를 하면서 단일 클론으로 변화하는 양상을 보였고, A형이 우세한 것으로 보아 집단 보균을 일으킨 유형 중 주 집단 클론은 A형이었음을 알 수 있었다. ESBL 생성 K. pneumoniae 보균 상태는 중환자실내에 입원기간 중에는 거의 지속되지만, 지역사회로 복귀하면 전부 해제되는 것으로 보인다. Purpose : The aims of this study included assessment of molecular-epidemiologic features during an outbreak of colonization of extended spectrum ${\beta}$-lactamase producing Klebsiella pneumoniae(ESBL-KPN) and re-evaluation of their colonized status one year later. Methods : Rectal swab cultures for ESBL-KPN from all hospitalized infants and newly admitted infants were obtained during the outbreak of colonization from July to December, 2000. The pattern of XbaI-digested chromosomal DNA of isolates were analyzed by pulsed-field gel electrophoresis. Weekly rectal swab cultures were obtained during the outbreak until patients were either discharged or decolonized. Patients discharged after being colonized had follow up stool cultures a year later. Results : A total of 80 patients(28.5 percent) were colonized. Of those, 53 whose pulsed-field gel electrophoresis(PFGE) was possible only once, were ESBL-KPN grouped into six cluster clones and 10 single clones : 28 patients(52.8 percent) were colonized with type A, the most common clone, followed by type B in 11 patients(20.8 percent). Of those 12 patients in whom serial PFGE was done more than twice, type A was predominant. Narrowed-down in strains occurred from types A, B, C, D and three single clones at initiation of the study into types A and type B after three months of strict infection control. Among 75 patients(93.7 percent) who were sent home after being colonized, 30 patients were re-called for stool cultures a year later : All of them were decolonized. Conclusion : This study demonstrates the importance of infection control as the diversity of ESBL-KPN strains could be narrowed into fewer strains. Colonization of ESBL-KPN could be reversed upon return to the community.

Antibiotic resistance, biochemical typing, and PFGE typing of Bifidobacterium strains commonly used in probiotic health foods

Feili Xu,Junping Wang,Yunchang Guo,Huawei Zeng,Ping Fu,Zhigang Li,Xiaoyan Pei,Xiumei Liu,Shuo Wang 한국식품과학회 2018 Food Science and Biotechnology Vol.27 No.2

This study firstly analyzed the antibiotic resistance, biochemical typing, and pulsed-field gel electrophoresis typing of 45 Bifidobacterium strains commonly used in health foods. Most strains were resistant to antibiotics but their antibiotic resistance rates were not high: Fos (56.52%), TET (43.48%), CRO (21.74%), AMC (15.22%), GEN (13.04%), RIF (10.87%), CHL (8.7%), CTX (6.52%), VAN (4.35%), and ERY (4.35%). The 45 strains could be divided into 14 pulsed-field gel electrophoresis types, of which the strain numbers of six pulsed-field gel electrophoresis types were more than one. All the Bifidobacterium strains could be divided into nine types by API50CHL biochemical identification. The same species displayed same biochemical typings, expect for B. animalis. Furthermore, the results confirmed that the same pulsed-field gel electrophoresis-type strains had closer antibiotic resistance patterns, and the same biochemicaltype strain also had similar antibiotic resistance patterns.

Multilocus Sequence Typing for Candida albicans Isolates from Candidemic Patients: Comparison with Southern Blot Hybridization and Pulsed-field Gel Electrophoresis Analysis

윤명하,신종희,이진솔,김수현,신명근,서순팔,양동욱 대한진단검사의학회 2011 Annals of Laboratory Medicine Vol.31 No.2

Background: We evaluated the efficacy of multilocus sequence typing (MLST) for assessing the genetic relationship among Candida albicans isolates from patients with candidemia in a hospital setting. Methods: A total of 45 C. albicans isolates from 21 patients with candidemia were analyzed. The MLST results were compared with results obtained by Southern blot hybridization (C1 fingerprinting) and pulsed-field gel electrophoresis (PFGE). PFGE analysis included karyotyping and restriction endonuclease analysis of genomic DNAs using BssHII (REAG-B) and SfiI (REAG-S). Results: The 45 isolates yielded 20 unique diploid sequence types (DSTs) by MLST, as well as 12 karyotypes, 15 REAG-B patterns, 13 REAG-S patterns, and 14 C1 fingerprinting types. Microevolution among intra-individual isolates was detected in 6, 5, 3, 5, and 7 sets of isolates by MLST (1 or 2 allelic differences), REAG-B, REAG-S, C1 fingerprinting, and a combination of all methods, respectively. Among 20 DSTs, 17 were unique, and 3 were found in more than 1 patient. The results of 2 DSTs obtained from 9 patient isolates were in agreement with REAG and C1 fingerprinting patterns. However, the remaining DST, which was shared by 2 patient isolates, showed 2 different PFGE and C1 fingerprinting patterns. In addition, 3 sets of isolates from different patients, which differed in only 1 or 2 alleles by MLST, also exhibited different PFGE or C1 fingerprinting patterns. Conclusions: MLST is highly discriminating among C. albicans isolates, but it may have some limitations in typing isolates from different patients, which may necessitate additional analysis using other techniques. Background: We evaluated the efficacy of multilocus sequence typing (MLST) for assessing the genetic relationship among Candida albicans isolates from patients with candidemia in a hospital setting. Methods: A total of 45 C. albicans isolates from 21 patients with candidemia were analyzed. The MLST results were compared with results obtained by Southern blot hybridization (C1 fingerprinting) and pulsed-field gel electrophoresis (PFGE). PFGE analysis included karyotyping and restriction endonuclease analysis of genomic DNAs using BssHII (REAG-B) and SfiI (REAG-S). Results: The 45 isolates yielded 20 unique diploid sequence types (DSTs) by MLST, as well as 12 karyotypes, 15 REAG-B patterns, 13 REAG-S patterns, and 14 C1 fingerprinting types. Microevolution among intra-individual isolates was detected in 6, 5, 3, 5, and 7 sets of isolates by MLST (1 or 2 allelic differences), REAG-B, REAG-S, C1 fingerprinting, and a combination of all methods, respectively. Among 20 DSTs, 17 were unique, and 3 were found in more than 1 patient. The results of 2 DSTs obtained from 9 patient isolates were in agreement with REAG and C1 fingerprinting patterns. However, the remaining DST, which was shared by 2 patient isolates, showed 2 different PFGE and C1 fingerprinting patterns. In addition, 3 sets of isolates from different patients, which differed in only 1 or 2 alleles by MLST, also exhibited different PFGE or C1 fingerprinting patterns. Conclusions: MLST is highly discriminating among C. albicans isolates, but it may have some limitations in typing isolates from different patients, which may necessitate additional analysis using other techniques.

Analysis of the genome of a Korean isolate of the Pieris rapae granulovirus enabled by its separation from total host genomic DNA by pulse-field gel electrophoresis

Yong Hun Jo,Bharat Bhusan Patnaik,Se Won Kang,Seunghan Oh,Dong Hyun Kim,Mi Young Noh,Gi Won Seo,Heon Cheon Jeong,Ju Young Noh,Hee Ju Hwang,Ji-Eun Jeong,Yeon Soo Han,Yong Seok Lee 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.10

Most traditional genome sequencing projects involving infectious viruses include culturing and purification of the virus. This can present difficulties as an analysis of multiple populations from multiple locations may be required to acquire sufficient amount of high-quality DNA for sequence analysis. The electrophoretic method provides a strategy whereby the genomic DNA sequences of the Korean isolate of Pieris rapae granulovirus (PiraGV-K) were analyzed by purifying it from host DNA by pulsed-field gel electrophoresis, thus simplifying sampling and labor time. The genomic DNA of infected P. rapae was embedded in agarose plugs, digested with a restriction nuclease and methylase, and pulsed-field gel electrophoresis (PFGE) was used to separate PiraGV-K DNA from the DNA of P. rapae, followed by mapping of fosmid clones of the separated viral DNA. The double-stranded circular genome of PiraGV-K encodes 120 open reading frames (ORFs), covering 92% of the sequenced genome. BLAST and ORF arrangement showed the presence of 78 homologs to other genes in the database. The mean overall amino acid identity of PiraGV-K ORFs was highest with the Chinese isolate of PiraGV (~99%), followed up with Choristoneura occidentalis ORFs at 58%. PiraGV-K ORFs were grouped, according to function, into 10 genes involved in transcription, 11 involved in replication, 25 structural protein genes, and 15 auxiliary genes. Genes for Chitinase (ORF 10) and cathepsin (ORF11), involved in the liquefaction of the host, were found in the genome. The recovery of PiraGV-K DNA genome by pulse-field electrophoretic separation from host genomic DNA had several advantages, compared with its isolation from particles harvested as virions or inclusions from the P. rapae host. We have sequenced and analyzed the 108,658 bp PiraGV-K genome purified by the pulsed field electrophoretic method. The method appears to be applicable to the analysis of genomes of large viruses. The chitinase, identified by PiraGV-K genome sequence, was functionally characterized by quantitative PCR, Western blot analysis, immunohistochemistry and transmission electron microscopy.

Antibiotic resistance, biochemical typing, and PFGE typing of Bifidobacterium strains commonly used in probiotic health foods

Xu, Feili,Wang, Junping,Guo, Yunchang,Fu, Ping,Zeng, Huawei,Li, Zhigang,Pei, Xiaoyan,Liu, Xiumei,Wang, Shuo 한국식품과학회 2018 Food Science and Biotechnology Vol.27 No.2

This study firstly analyzed the antibiotic resistance, biochemical typing, and pulsed-field gel electrophoresis typing of 45 Bifidobacterium strains commonly used in health foods. Most strains were resistant to antibiotics but their antibiotic resistance rates were not high: Fos (56.52%), TET (43.48%), CRO (21.74%), AMC (15.22%), GEN (13.04%), RIF (10.87%), CHL (8.7%), CTX (6.52%), VAN (4.35%), and ERY (4.35%). The 45 strains could be divided into 14 pulsed-field gel electrophoresis types, of which the strain numbers of six pulsed-field gel electrophoresis types were more than one. All the Bifidobacterium strains could be divided into nine types by API50CHL biochemical identification. The same species displayed same biochemical typings, expect for B. animalis. Furthermore, the results confirmed that the same pulsed-field gel electrophoresis-type strains had closer antibiotic resistance patterns, and the same biochemical-type strain also had similar antibiotic resistance patterns.

Extended-Spectrum β-Lactamase 생성 Klebsiella pneumoniae 감염의 Pulsed-Field Gel Electrophoresis를 이용한 역학적 분석

이경원,정윤섭,서설송,신희봉,권오헌,고신옥,정석훈 대한감염학회 1996 감염 Vol.28 No.5

목 적 : 근래 제 3세대 cephaolsporin 내성 K. pneumoniae의 분리율의 증가가 보고된 바 있다. 본 연구에서는 세브란스 병원 환자에서의 cefotaxime 내성 K. pneumoniae의 분리 추이와 그 내성 원인이 ESBL에 의한 것인지를 규명하고자 하였다. 또한 ESBL 생성 K. pneumoniae의 염색체 DNA를 제한효소로 절단하여 PFGE로 그 양상을 비교하여, 이들 세균 감염의 역학적 지견을 넓히고자 하였다. 방 법 : 1988-1995년에 세브란스 병원 환자에서 분리되 K. pneunoniae의 cefotaxime 내성을 NCCLS디스크 확산법으로 시험하였다. Cefotaxime에 중간이나 내성인 균주 118주를 대상으로 cefotaxiem, ceftazidime, aztreonam 및 amoxicillin/clavulanic acid 디스크를 사용하여 double disk synergy를 시험하였다. 또한 ESBL 생성균주의 염색체 DNA를 XbaI으로 절단 후 PFGE 양상을 분석하였다. 결 과 : Cefotaxime에 감수성인 균주는 1988년에 78%였으나 점차 감소하여 1995년에는 46%였다. 검체에 따른 cefotaxime에 대한 감수성 비율은 별 차이가 없었으나, 각담과 요에서 흔히 분리되었다. 내성율은 중환자실 환자에서 분리된 균주가 일반병실이나 외래환자에서 분리된 균주에 비하여 높았다. 1994년에 분리된 cefotaxime에 중간 또는 내성인 K. pneumoniae 70주 중 54주 (77%) 와 1995-1996년에 분리된 48주 중 41주 (85%) 는 ESBL을 생성하였다. ESBL 생성 K. pneumoniae 88주의 염색체 DNA의 XbaI에 의한 절단 양상을 PFGE로 비교한 바, 28가지 형으로 분류되었다. 1994년에 분리된 균주 중에는 A형이, 1995-1996년에 분리된 균주 중엔 D형이 많았으며, 이들은 대부분 중환자실 환자에서 분리되었다. 결 론 : 3차병원에서 분리되는 K. pneumoniae중에는 제 3세대 cephalosporin에 중간 또는 내성인 균주의 비율이 증가하고 있고, 그 중 대부분은 ESBL을 생성하는데, 이들은 중환자실 환자에서 흔히 분리되며, 염색체 DNA형이 같거나 비슷한 예가 많아 소수 클론에서 유래된 세균으로 판단되었으며, 이들 세균의 전파방지를 위한 조치가 필요하며, 제 3세대 cephalosporin에 감수성이 중간인 균주도 이 항균제에 내성인 것으로 간주하여 치료하여야 할 것으로 사료되었다. Background : Third generation cephalosporins were potent antimicrobial agents against gram-negative bacilli, however, increased resistance to these drugs has been noted recently. This study was to determine the prevalence of cefotaxime resistance and extended-spectrum β-lactamase (ESBL) in Klebsiella pneumoniae, and to determine for the existence of hospital infection by this organism. Methods : Cefotaxime resistance was tested by NCCLS disk diffusion method from 1988 to 1995 in Severance Hospital. Among the cefotaxime-intermediate or resistant strains, 118 were tested for ESBL production by double disk synergy test using cefotaxime, ceftazidime, aztreonam, and amoxicillin/clavulanic acid disks. The patterns of XbaI-digested chromosomal DNA of ESBL-producing K. pneumoniae isolates were analyzed by pulsed-field gel electrophoresis. Results : Cefotaxime-susceptible rates decreased from 78% to 46% in K. pneumoniae during the recent 8 years. The isolates from ICU patients showed higher resistance rates than those from general wards or outpatient clinics. Among the strains with intermediate or resistance to cefotaxime, 77% were isolated in the period of March to August, 1994 and 85% in October, 1995-January, 1996 were double disk synergy test-positive. ESBL-producing K. pneumoniae were grouped into 28 chromosomal DNA patterns by Xbal digestion. Type A were the most common in 1994 and type D in 1995 to 1996, and these were prevalent among the strains isolated from ICU patients. Conclusion : It is concluded that proportion of ESBL-producing K. pneumoniae strains has been increasing. They are more frequently isolated from the specimens of intensive care unit patients, and considered to be derived from a few clones. In additon, third generation cephalosporin-intermediate strains should be considered to be clinically resistant to the drugs.