Characters of Extracellular β-Lactamase Obtained from a Strain of Streptomyces sp.

Moon, Sang Beom,Lee, Kye Joon 한국산업미생물학회 1991 한국미생물·생명공학회지 Vol.19 No.5

A strain of Streptomyces sp. isolated from soil was found to produce extra-cellular β-lactamase associated partially to the cell growth. The β-lactamase was purified from the culture supernatant through ammonium sulfate fractionation, ion-exchange chromatographies and gel filtration. The final purification fold and recovery yield were 57 and 6.2%, respectively. Molecular weight of the β-lactamase was estimated to be about 67,000 by SDS-polyacrylamide gel electrophoresis. The optimal reaction condition was at pH 7∼8 and at 35∼45℃. The K_m and V_max values of the enzyme for penicillin G were estimated to be 3 mM and 1.3×10 exp(3) uM/min/㎎ protein, respectively. The purified β-lactamase was classified to the class A enzyme hydrolyzing only penicillin.

만성 편도염의 세균학적 고찰 및 Beta Lactamase의 측정

전병훈,김승곤,김동석,서일원 인제대학교 1991 仁濟醫學 Vol.12 No.3

급성 편도염의 원인균은 Group A β-hemolytic streptococcus에 의한 것으로 Penicillin이 특효로 여겨져 널리 사용되고 있으나 최근 Penicillin의 투여 후에도 만성 또는 재발하는 경향을 보여 Penicillin의 내성균을 규명하기 위하여 만성 편도염 환자의 편도조직에서 세균총을 배양하여 세균학적 검사와 β-lactamase를 측정한 결과 β-lactamase를 생산하는 균주들에 의한 것으로 추정할 수 있었다. Chronic tonsillitis has long been one of the most common diseases in the otolaryngologic field. The failure of Penicillin to eradicate group A β-hemolytic streptococci is a growing problem. The shift in the population of microflora with selection of β-lactamase producing strain is regarded as a major cause. In this study we have done bacteriologic cultures and evaluated the prevalences of β -lactamase producing organisms in 32 excised tonsils from patients with recurrent tonsillitis and the results were following : 1.Total 110 strains were isolated; Neisserla 30(27.3%), Corynebacterium sp. 13(11.9%), Staphylococcus aureus 11(10%), and H. influenza 9(8.2%). 2.All tonsils showed mixed infection with average 3.4 strain per tonsil 3.β-lactamase producing organisms were detected from 19 tonsils(59.3%) and the strains were Staphylococcus aureus 9, Neisserla sp. 7, H.influenza 7, and Branhamella sp. 2.

Metallo-β-lactamase 생성 녹농균에서 Class 1 Integron의 유전형 분석

성지연,구선회,권계철,박종우,고지선,신소연,송정훈 대한임상미생물학회 2009 Annals of clinical microbiology Vol.12 No.1

Background: The genes of metallo-β-lactamase (MBL), a powerful carbapenemase, are carried as a part of the mobile gene cassettes inserted into integrons playing an important role in rapid dissemination of antibiotic resistance genes among bacterial isolates. In this study, we investigated carbapenemase genes and class 1 integrons integrated into the gene cassettes in imipenem-non susceptible P. aeruginosa. Methods: From July 2006 to March 2008, 81 consecutive, non-duplicate, imipenem-non susceptible P. aeruginosa were isolated at Chungnam National University Hospital in Chungcheong province of Korea. The modified Hodge and double disk synergy tests were conducted for the screening of carbapenemase and MBL production, respectively, and PCR and DNA sequencing were performed for the detection of carbapenemase genes and class 1 integron gene cassettes. We also employed the repetitive element sequence- based (Rep)-PCR method for an epidemiologic study. Results: MBLs were detected in 13.6% (11/81) of imipenem-non susceptible P. aeruginosa. Ten isolates were found to carry blaIMP-1, whereas 1 isolate was found to carry a blaVIM-2. All of the IMP-1-producing strains harbored 4.0 kb class 1 integron containing chloramphenicol, aminoglycoside, and β-lactamresistant genes. However, blaIMP-1 was not detected at class 1 integron. A 2.5 kb class 1 integron harboring blaVIM-2 was detected in a VIIM-2- producing strain. One identical pattern was observed in ten IMP-1 producing strains. Conclusion: IMP-1 producing P. aeruginosa strains are currently distributed throughout Chungcheong province of Korea. In particular, all of the strains harbored class 1 integrons containing variant antibiotic resistance gene cassettes.

Bacillus sp. J105 유래 β-lactamase 유전자의 cloning 및 E. coli 내에서의 발현 분석

강원대(Won Dae Kang),임학섭(Hak Seob Lim),서민정(Min Jeong Seo),김민정(Min Jeong Kim),이혜현(Hye Hyeon Lee),조경순(Kyeong Soon Cho),강병원(Byoung Won Kang),서권일(Kwon Il Seo),최영현(Yung Hyun Choi),정영기(Yong Kee Jeong) 한국생명과학회 2008 생명과학회지 Vol.18 No.11

β-Lactam계 항생물질에 강한 내성을 가지는 균주 Bacillus sp. J105가 생산하는 β-lactamase의 유전자를 E. coli DH5α에 cloning하였다. Cosmid vector pLAFR3을 이용하여, Sau3AI으로 부분 분해한 chromosomal DNA와 BamHI으로 처리한 pLAFR3을 ligation하였다. In vitro packaging kit를 사용하여 E. coli에 형질도입 하였으며 β-lactamase양성 clone주를 획득하였다. 이 recombinant plasmid (β-lac+)를 pACYC184 (4.2 kb) vector를 사용하여 subcloning 하여 최종 β-lactamase의 활성이 있는 6.4 kb 단편이 포함된 pKL11-Δ4.6을 제작하였다. 이 단편을 DNA 염기서열을 분석한 결과 309개의 아미노산으로 구성된 β-lactamase를 코딩하는 927 bp를 포함하고 있었다. 클로닝된 β-lactamase 유전자의 upstream을 포함하는 170 bp의 염기서열을 분석한 결과, B. thuringinesis와 B. cereus 유래의 β-lactamase 유전자의 upstream 부위와 97%의 일치를 보였다. 본 연구에서 클로닝된 β-lactamase의 아미노산을 서열을 NCBI BLAST program을 이용하여 분석해 본 결과 B. thuringinesis와 B. cereus의 β-lactamase와 각각 97%와 94%의 일치를 보였다. 또한 계통도 분석 결과 역시 본 연구에서 클로닝된 β-lactamase의 아미노산을 서열은 B. thuringinesis와 B. cereus와 유전학적으로 아주 밀접한 관계를 보여주었다. 이 pKL11-Δ4.6를 E. coli에서 형질전환 시켜 발현 양상을 조사해 본 결과 β-lactamase의 secretion efficiency는 약 4~5%였다. E. coli의 세포 내 단백질로부터 β-lactamase를 정제하여 분자량을 확인한 결과 31 kDa로 wild type의 분자량과 일치함을 확인하였다. The β-lactamase gene was cloned into E. coli DH5α from Bacillus sp. J105 with strong resistance against β-lactam antibiotics. The chromosomal DNA was partially digested with Sau3AI and ligated to BamHI digested pLAFR3. β-Lactamase positive clones were obtained by using in vitro packaging kit. The pKL11-Δ4.6 with β-lactamase activity was obtained by subcloning of the recombinant plasmid (β-lac +). The 6.5 kb fragment in the subcloned plasmid was sequenced. The DNA fragment that contains the β-lactamase gene encodes 309 amino acids. The 0.17 kb upstream region was similar to those of B. thuringinesis and B. cereus with 97% identity. The deduced amino acids sequence was also similar to those of β-lactamase from B. thuringinesis and B. cereus with 97% and 94% identity, respectively. The phylogenetic tree also showed the relationships of the β-lactamase gene of Bacillus sp. J105 to genetically related that of other Bacillus strains. Analysis of expression pattern of the pKL11-Δ4.6 in E. coli, revealed that the secretion efficiency of β-lactamase was 4~5% and the molecular weight was as same as that of original β-lactamase (31 kDa) from Bacillus sp. J105.

Cefatrizine과 Clavulanic acid 병합제의 항균작용의 향상

최성학,김계원,김지영,김원배,이경원,정윤섭 대한화학요법학회 1998 대한화학요법학회지 Vol.16 No.3

배경 : β-lactam계 항균제 내성의 주된 원인은 β-lactam 항균계를 가수분해하는 β-lactamase에 기인한다. 경구용 cephalosporin인 cefatrizine (CTRZ)과 β-lactamase의 inhibitor인 clavulanic acid (CV)의 병합제 투여는 β-lactamase 생성 내성균에 유효하다. 본 연구는 β-lactamase에 대한 CV의 저해 특성을 조사하여 CTRZ와 CV의 병합제 투여시 상승효과를 얻을 수 있는 제형의 가능성에 대하며 검토하였다. 방법 : CV와 CTRZ의 β-lactamase 저해활성은 IC_(50)으로 측정하였고, CV의 노출에 따라 CTRZ의 항균활성에 미치는 영향은 postantibiotic effects (PAE), post, β-lactamase inhbitor effects(PLIE), bactericidal effectsin vitro simulation bactericidal effects로 평가하였다. 결과 : CV는 extended spectrum β-lactamase class A β-lactamase에 강한 저해작용을 나타내었다. CTRZ/CV에 2시간 노출시킨 E. coli EB13, K. pneumoniae EB40과 M. catarrhalis 6012에서 PAE와 PLIE가 인정되었고 PLIE가 PAE보다 크게 나타났다. CTRZ/CV의 살균력은 CV에 노출되지 않은 세균보다 CV에 노출된 세균에서 우수한 결과를 나타내었다. E. coli EB13에 대한 In vitro simulation bactericidal effects에서 CTRZ 250 mg투여하고 2시간 후에 CTRZ 250mg 투여 시 예상되는 인체의 혈중농도 profile을 적용시켰을 때 CTRZ 500 mg과 CV 250 mg 투여 시 예상되는 인체의 혈중농도 profile을 적용시킨 경우의 항균활성 개선 효과가 관찰되었다. 결론 : CTRZ의 유효혈중농도를 지속적으로 유지할 수 있는 CTRZ/CV 방출 제어 복합항생제를 고안한다면 β-lactamase 생산에 의하여 내성을 획득한 세균 감염증에 대하여 우수한 치료효과를 기대할 수 있는 항균제의 개발이 가능할 것으로 생각된다. Background : β-lactamase play an important role in bacterial resistance against β-lactamase antibiotics. The resistant strains due to the β-lactamases including extended spectrum β-lactamase were susceptible to a combination of cefatrizme (CTRZ) and clavulanic acid (CV), a oral cephalosporin and a β-lactamase inhibitor. We studied properties of β-lactamase inhibition by CV and proposed a concept that the controlled dosing of CTRZ and CV combination can produce more effective synergistic antibacterial activity. Methods : Inhibition activities of CTRZ and CV against various plactamases were determined by IC?? and effects of CV exposure on antibacterial activity of CTRZ and CV combination were estimated by postantibiotic effects (PAEs), post β-lactamase inhibitor effects (PLIEs), bactericidal effects and in vitro simulation bactericidal effects. Results : The class A and extended spectrum plactamases were inhibited by CV. PAEs and PLIES were shown to Escherichia coli EB13, Klebsiella pneumoniae EB40 and Moraxella catarrhalis 6012. The PLIEs were prolonged more than the corresponding PAEs. CV-exposed strains were rapidly killed and delayed in regrowth compared to the unexposed strains. The sequential dosing of CTRZ (250 mg) two hours after CTRZ (250 mg) plus CV (250 mg) enhanced antibacterial activity than simultaneous dosing of CTRZ (500 mg) and CV (250 mg) against E. Coli EB13 in in vitro simulation bactericidal effects. Conclusion : When a combination of CTRZ and CV was used in human, the controlled-release combination to maintain long CTRZ plasma levels above a cntical concentration was useful for treatment of β-lactamase producing bacterial infections.

베타-락타메이즈 저해제를 생성하는 방선균의 분리

이상희 명지대학교 자연과학연구소 2005 자연과학논문집 Vol.24 No.-

β-lactamase inhibitor를 생성하는 균주를 선별하기 위해서 세계각지의 토양에서 분리되어진 방선균으로부터 β-lactam 항생제(penicillin G, 100 U/㎖), bactopenase(500 U/㎖) 및 지시균으로써 Staphylococcus aureus ATCC6538P를 첨가한 배지를 이용하여 저해환을 측정하였다. 분리 되어진 6,249개 방선균으로부터 92균주를 일차 선별하였으며, 이들을 ampicillin(100 ㎍/㎖)과 bla_(TEM-la) gene을 함유하는 Escherichia coli DH5α를 이용하여 저해환을 측정하고, 그 결과를 일차 선별 결과와 비교함으로써 지시균에 대해 가장 큰 저해능을 나타내는 6균주를 최종 선별하였다. 선발된 6 균주를 β-lactamase inhibitor 또는 β-lactamase inhibitor protein을 생성하는 균주로 밝혀진 균주들(Streptomyces cluvuligerus 또는 S. exfoliatus)과 비교실험을 통해서 선발된 6 균주는 출현하는 항생제 내성 임상균주에 대해 작용하는 β-lactamase inhibitor를 생성하는 것으로 확인 되었다. In order to screen Streptomyces spp. producing β-lactamase inhibitors from 6,249 Streptomyces spp. isolated from several worldwide soil samples, analyses of inhibition zones were performed using penicillin G (100 ㎍/㎖), bactopenase (500 U/㎖), and Staphylococcus aureus ATTCC6538P. The first screening showed that 92 isolates produced β-lactamase inhibitors (BLIs). By the final screening using ampicillin (100 ㎍/㎖) and Escherichia coli DH5a harboring bla_(TEM-la) gene (500 U/㎖), six inhibitor-producing isolates were selected. The comparison experiments using Streptomyces cluvuligerus and S. exfoliatus producing BLI and β-lactamase inhibitor protein (BLIP), respectively confirmed that the six strains produced BLI (not BLIP) anainst clinical isolates presently detected and showing β-lactam antibiotic resistance.

병원 내 환자에서 분리한 Klebsiella pneumoniae에서 광범위 β-lactamase의 분자생물학적 특성

위성현,김수영,김상일,김양리,유진홍,신완식,강문원 대한병원감염관리학회 2003 의료관련감염관리 Vol.8 No.2

성인에서 Haemophilus influenzae 감염증의 임상양상과 미생물학적 특성

이종섭,황병연,정희진,김우주,박승철,이도현,이창규,신종희,황규잠,이영희 대한화학요법학회 2000 대한화학요법학회지 Vol.18 No.2

목적 : H. influenzae는 성인에서 흔히 호흡기 감염을 일으키는 균주로, 최근 ampicillin 내성 β-lactamase 생성 균주들이 지속적인 증가를 보이고 있어, 이에 국내 성인에서 H. influenzae에 의한 감염의 임상양상과 분리된 균주들의 미생물학적 특성을 조사하고자 하였다. 방법 : 1998년 3월부터 1999년 9월까지 고려대 구로병원에 내원한 환자중에서 H. influenzae가 분리된 68명을 대상으로 임상양상을 조사하고 항균제 감수성 검사를 시행하였다. 분리된 33주에 대해서 혈청형, 생물형, 세포외막 단백분석을 시행하였고, β-lactamase 생성여부에 따른 감염 환자군간 임상상 및 예후의 차이를 비교하였다. 결과 : 환자의 평균연령은 68세였고, 지역사회 획득감염이 전체의 85%를 차지하였다. 폐렴과 기관지염과 같은 하기도 감염이 전체환자의 75%를 차지하였고 분리 검체 역시 객담이 84%로 가장 많았다. 환자의 71%가 기저질환을 가지고 있었으며, 그 중에 만성 폐쇄성 폐질환, 심혈관 질환, 악성종양, 당뇨병이 대부분이었다. 혈청형으로는 nontypeable 형이 68%로 가장 많았고 b형은 한 예도 없었다. β-lactamase 생성율은 63.2%였고, β-lactamase 양성균주의 항균제 내성율이 음성균주에 비하여 높았으나 β-lactamase 생성 여부에 관계없이 cefotaxime, azithromycin, ciprofloxacin에 대하여 100%의 감수성을 나타내었고 cefaclor, cefuroxime에 대해서는 10% 이하의 낮은 내성율을 보여 이들 항균제들이 치료에 효과적일 것으로 사료된다. 세포외막 단백분석에서는 C형과 D형이 전체의 64%로 가장 많았으며, 혈청형과의 유의한 연관성은 보이지 않았다. 결론 : 성인에서 H. influenzae는 주로 만성 기저질환자에서 급성 호흡기감염 또는 만성 폐쇄성 폐질환의 급성 악화를 유발한다. 기존의 보고에 비해 β-lactamase 양성율이 증가하였고, 이에 따른 항균제 내성율도 증가하여 항균제의 선택에 유의하여야 할 것으로 생각된다. 세포외막 단백분석상 특정한 유형의 균주에 의해 감염이 집중적으로 발생하고 있어 향후 역학 조사의 도구로 이용할 수 있겠다. Purpose : H. influenzae is one of the common causative microorganisms of respiratory tract infections in adults. Recently. β-lactamase producing, ampicillin-resistant H. infiuenzae has become worldwide problem as well as in Korea. We studied to investigate the clinical features and microbiologic characteristics of H. infiuenzae infections in adults. Materials & Methods : From March 1998 to September 1999, 68 patients were enrolled in this study. Clinical features of H. infiuenzae infections were investigated, 68 isolates were subjected to the β-lactamase test, and 33 isolates were used for serotyping. biotyping, antibiotic susceptibility and outer membrane protein (OMP) analysis. Results : Mean age of study patients was 68 years-old and the acquisition rate in community was 85%. Pneumonia and bronchitis were the most common type of infection such as 80%, sputum was the most common clinical specimen for H. mQuenzae isolation. 70% of patients had chronic underlying diseases. Non-typeable strains were 68% and β-lactamase producing rate was 63%. There was no difference in the clinical features and prognosis of H. influenzae infection between the β-lactamase positivie and -negative groups. The susceptibility of cefotaxime, azithromycin, and ciprofloxacin was 100% and that of cefaclor, cefuroxime was under 10% in spite of β-lactamase positivity. In OMP analysis, C and D types were dominant (64%). Conclusion : H. influenzae caused acute respiratory infections in adults with chronic underlying disorders. This study shows higher β-lactamase producing rate and antibiotic-resistance rate than that were reported previously. OMP analysis shows that two major types of strains cause clinical infections intensively.

家畜 및 原乳에서 分離한 satphylococcus aureus의 β-lactamase 産生과 藥劑 感受性

Chul Heui Jung(鄭哲喜),Ho Jo Kang(姜鎬祚) 한국예방수의학회 1993 예방수의학회지 Vol.17 No.3

The present study was carried out for the purpose of investigating the β-lactamase production and drug resistance of Staphylococcus aureus isolated from bulk milk and domestic animals. Frequency of β-lactamase producing strains was 94.4% of bulk milk isolates, 35.4% of chicken isolates, 26.9% of cattle isolates and 5.9% of pig isolates in 280 S. aureus isolates. The 90% MIC of kanamycin was more than 100㎍/ml but was 6.29㎍/ml to methicillin and 1.56㎍/ml to oxacillin. Most of 280 isolates were sensitive to cephalexin, methicillin and oxacillin but 23.9%-55.4% of the strains were resistant to kanamycin, ampicillin and penicillin. The β-lactamase nonproducers were more sensitive than producers to penicillin and ampicillin on the basis of MICs in arithmetic mean, but there were no differences in sensitivity of these groups to cephalexin, methicillin, oxacillin and kanamycin. The most frequently encountered drug resistant patterns were AmPc in double patterns and AmKmPc in triple patterns. The 87.1% of β-lactamase producing strains were ampicillin resistant but no β-lactamase nonproducing strains were resistant. The resistant rates of β-lactamase producers generally were higher than those of β-lactamase nonproducers to all antibiotics. Most of β-lactamase producing strains harborred plasmids of 25kb or 48kb. The 48kb plasmid was suspected as the penicillin-resistant plasmid after plasmid curing experiment.

Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca와 Proteus mirabilis 에서 Boronic Acid Disk를 이용한 Extended-spectrum β-lactamase와 AmpC β-lactamase 검출

박순덕,어영,장인호,권오건,윤갑준,김효열 대한임상미생물학회 2009 Annals of clinical microbiology Vol.12 No.1

Background: Accurate detection of organisms producing extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase is very important for treatment of patients. However, unlike the ESBL confirmatory test, there are no guidelines for detection of organisms producing AmpC β-lactamase. We evaluated a detection method using boronic acid (BA) for ESBL and AmpC β-lactamase. Methods: Clinical isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis showing intermediate resistance or resistance to cefoxitin (FOX) or positive for ESBL were tested. A ≥5 mm increase in zone diameter of ceftazidime/ clavulanic acid/BA (CAZ/CA/BA) and/or cefotaxime/ clavulanic acid/BA (CTX/CA/BA) versus CAZ/BA and/or CTX /BA was considered positive for ESBL. Likewise, a ≥5 mm increase in zone diameter of FOX/BA and/or cefotetan/BA (CTT/BA) versus FOX and/or CTT alone was considered positive for AmpC β-lactamase. Results: Among 622 clinical isolates, ESBL positive rates by the CLSI ESBL confirmatory test or by the BA method were 18.1% or 18.4% for E. coli, 38.3% or 40.4% for K. pneumoniae, 8.7% or 8.7% for K. oxytoca, and 14.8% or 14.8% for P. mirabilis, respectively. AmpC β-lactamase positive rates using the BA method were 3.7% for E. coli, 33.3% for K. pneumoniae, 0% for K. oxytoca, and 7.4% for P. mirabilis. The detection rates of coproducing ESBL and AmpC β-lactamase were 2.4% in E. coli 27.1% in K. pneumoniae, and 3.7% in P. mirabilis. Conclusion: The ESBL confirmatory method using BA was found to enhance the detection of ESBLs, even when potentially masked by AmpC β-lactamase.