충청지역병원에서 분리된 Extended-Spectrum β-Lactamase 생성 대장균과 폐렴간균의 bla 유전형 및 분자역학적 분석

육근돌,양병선,박진숙,Yook, Keun Dol,Yang, Byoung Seon,Park, Jin Sook 한국미생물학회 2014 미생물학회지 Vol.50 No.2

충청지역병원에서 ESBL생성 장내세균 122균주를 분리하여 Clinical and Laboratory Standards Institute (CLSI) 방법에 따라 cefotaxime과 cefotaxime/clavulanate를 이용한 combination disk test (CDT)에 의하여 항균제 감수성을 조사하고, 특이 유전자를 대상으로 multiplex PCR을 실시하여 유전형을 검출하였으며 enterobacterial repetitive intergenic consensus (ERIC)-PCR에 의해 분자 역학조사를 실시하였다. ESBL 생성 Escherichia coli 76균주와 Klebsiella pneumoniae 46균주를 CDT로 확인한 결과, ESBL 생성 E. coli의 경우 96.1%가 양성을 나타내었으며 K. pneumoniae의 경우 93.4%가 양성을 나타냈다. Multiplex PCR 결과, E. coli의 경우 CTX-M-2형이 60.5% 양성으로 나타났으며 K. pneumoniae의 경우 VEB-1형이 56.5%가 양성으로 나타났다. ERIC-PCR을 실시한 결과 E coli는 분리지역에 따라 5개의 cluster을 형성하였고 K. pneumoniae는 4개의 cluster을 형성하였다. ESBL생성 장내세균의 유전형은 임상에서 분리한 균주의 감별과 검출에 유용하였으며 ERIC-PCR의 결과는 분리지역에 따라 cluster을 형성하여 지역 간 감염 감시체계 확립에 도움이 될 것으로 사료된다. A total of 122 ESBL-producing intestinal bacteria were collected from regional hospitals in the Chungcheong area. Combination disk test (CDT) was performed for antimaicrobial susceptability using cefotaxime and cefotaxime/clavulanate according to Clinical Laboratory Standard Institute (CLSI). Mutiplex PCR using specific primers was performed for a detection of ESBL-genotypes and enterobacterial repetitive intergenic consensus (ERIC)-PCR was carried out for the tracking of molecular epidemiology. In the confirmation test using CDT, 73 out of 76 (96.1%) ESBL-producing Escherichia coli and 43 out of 46 (93.4%) ESBL-producing Klebsiella pnemoniae were positive. In the multiplex PCR, 60.5% of E. coil were positive for CTX-M-2 type gene and 56.5% of K. pneumoniae were positive for VEB -1 type gene. In the ERIC-PCR, E. coil isolates formed 5 clusters and K. pneumoniae isolates were grouped into 4 clusters depending on region. Genotypes of clinical isolates are useful for detection and differentiation of ESBL producing intestinal bacteria. The ERIC-PCR method is thought to be helpful for establishing a regional surveillance system for infection due to its formation of different clusters depending on region.

Klebsiella pneumoniae균의 바이오 필름 형성과 활성에 대한병두황진 추출물의 효과

육근돌,하나영 대한임상검사과학회 2018 대한임상검사과학회지(KJCLS) Vol.50 No.4

The emergence of biofilms have generated urgent alarm in clinical and medicine manufacturing fields engaged in the search for novel antimicrobials from ethno-medicinal plants. The National Institutes of Health (NIH) has estimated that 70% of all microbial infections in the world are associated with biofilms. In addition, the emergence of strains resistant to conventional antibiotics has become a serious threat to global public health. Therefore, finding alternative medicines is a major issue in the field of integrative medicine. In this study, four different herb extracts were screened for biofilm formation and the activities of Klebsiella pneumoniae. Of them, Scutellaria scordifolia Fisch. ex Schrank extracts had inhibitory effects on bacterial growth and biofilm formation. The Scutellaia scordifolia Fisch. ex Schrank extracts did not cause any cytotoxicity to L929 cells. The growth of K. pneumoniae was inhibited compared to other comparators in the experimental group containing Scutellaia scordifolia Fisch. ex Schrank. In a group of experiments with plant extracts, a maximum of 60 times the level of living bacteria was confirmed compared to the controls without the addition of the Scutellaia scordifolia Fisch. ex Schrank extracts. In a group of experiments with a significantly lower level of fluorescence extraction, differential interference contrast imaging showed that the number of K. pneumonae was reduced. These results suggest that extracts of this plant be applied as antimicrobial agents against K. pneumoniae, particularly in biofilm forms. 주술적 의료로 이용되는 식물에서 새로운 항미생물질을 찾는있는 중에 바이오필름의 출현은 임상, 의학 제조업 분야에서 다급한 신호를 보내주고 있는 것이다. 국립보건원(NIH)은 세계 모든 미생물 감염의 70%가 바이오필름과 연관되어 있다고 추정하고 있으며, 기존의 항생제에 내성이 있는 변종의 출현은 전세계공중 보건에 심각한 위협이 되고 있다. 따라서 대체의학 물질을찾아 내는 것은 통합(동서양) 의학의 중요한 화두이다. 이 연구에서 Klebsiella pneumoniae의 바이오 필름 형성과 활성화에 대한 네 가지 허브 추출물을 스크린 하였다. 본 실험에 사용된 식물추출액 중에 Scutellaria scordifolia Fisch. ex Schrank 추출물이 박테리아의 성장과 바이오 필름 형성에 억제 효과를 보여 주었다. Scutellaria scordifolia Fisch. ex Schrank 추출물이 L929세포에 대한 세포독성을 띄지 않음을 확인 하였다. Scutellaria을 첨가한 실험군에서 다른 대조군에 비해 K. pneumoniae의 성장이억제되었다. Scutellaria scordifolia Fisch. ex Schrank 추출액이 첨가되지 않은 대조군과 비교하면, 식물 추출액이 첨가된 실험군에서는 생균수가 최대 약 60배 억제되었음을 확인 하였다. Scutellaria scordifolia Fisch. ex Schrank 추출물이 포함된 실험 군에서 K. pneumoniae 균의 개체수가 줄어들어 있음을differential interference contrast (DIC)의 이미지를 통해서도확인하였으며, 형광세기의 비교분석을 통해 음성대조군에 비해추출물이 포함된 실험군에서 형광세기가 통계학적으로 유의하게 낮아져 있는 것을 확인하였다. 이러한 결과는 이 식물 추출물이 K. pneumoniae의, 특히 바이오필름 형성에서 항균제로서 적용될 수 있음을 암시해 주는 것이다.

대학생 잠복결핵 감염의 진단

육근돌 ( Keun-dol Yook ),양병선 ( Byoung-seon Yang ) 대한임상검사과학회 2016 대한임상검사과학회지(KJCLS) Vol.48 No.3

결핵이란 결핵균에 의한 만성 감염질환으로 결핵균이 포함된 비말액을 통해 공기감염을 일으킨다. 대부분의 결핵감염자는 전염력이 없는 잠복감염상태만 유지하나 10%의 감염자 중 절반은 감염 후1∼2년 안에 발병하게 된다. 결핵감염자와 접촉이 있는 학생 74명을 대상으로 결핵 감염자 접촉관련 사전 조사 및 흉부 X-선 검사, TST 검사 및 IGRA 검사를 실시하였다. 1차 TST 검사에서 양성자는 9명, 음성은 65명으로 나타났으며, 음성 판독자는 2차 TST검사를, 양성 판독자는 IGRA 검사를 실시하였다. 1차 TST 양성자 9명중 IGRA 검사에서 3명이 양성자로 나와 잠복 결핵감염 치료를 실시하였다. 1차 TST결과 음성자에 대한 재확인 TST 검사는 1차와 차이가 없었다. 결론적으로 잠복결핵감염율은 74명중 3명으로 4.05%로 나타났고, 잠복결핵감염자 색출을 위해서 다른 그룹에 대한 조사도 필요하다. Tuberculosis (TB) is caused by a chronic infectious agent known as Mycobacterium tuberculosis. It is transmitted in airborne particles, called droplet nuclei which was generated by cough, sneeze, shout, or sing of persons who have TB disease. Most infections of TB do not have symptoms, well known as latent tuberculosis infection (LTBI). However, about 10% of LTBI progress to active disease a one or two years after infection. To investigate the LTBI rate of college students who were in contacted with TB patients, we performed chest X-ray, tuberculin skin test (TST) and Interferon-gamma release assay (IGRA) to 74 college students. At a results, 65 students were showed negative and 9 students positive results at chest X-ray and 1st TST test. When confirmed the 65 students who were showed negative by 2st TST, the results showed correctly. But, 9 students who were showed positive results on chest X-ray and 1st TST by IGRA, the only 3 students (4.05%) showed positive results. In conclusion, the LTBI rate in this study showed 4.05% (3/74) and we suggest to investigate other students LTBI rate for decreasing tuberculosis.

폐렴 막대균에 대한 현삼(Scrophularia ningpoensis hemsl) 추출액의 항균력과 세포독성 시험

육근돌(Keun-Dol Yook) 한국컴퓨터정보학회 2016 韓國컴퓨터情報學會論文誌 Vol.21 No.5

Scrophularia ningpoensis hemsl has been traditionally used in China and Vietnam for treatment of bacteria, atopy, pimple, tonsillitis, angina and encephalitis for a long time. The main objectives of this study were to evaluate the antibacterial activity of the Scrophularia ningpoensis hemsl extract on biofilm formation of Klebsiella pneumoniae. Antibacterial activity was conducted using disc diffusion assay and minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) were determined using the broth micro dilution method in accordance to Clinical and Laboratory Standards Institute guidelines(CLSI). Furthermore, cytotoxicity on L929 were assessed using animal cell culture for the proliferation test(MTT cell assay) and the biofilm forming capacity of the K. pneumoniae were determined using the colony forming unit (CFU) assay. The extract exhibited considerable antibacterial activity. K. pneumoniae was susceptible to the extract with the MIC and MBC of 0.1875 and 1.5 ㎎/㎖ respectively. Cytoxicity test in L929 showed no sign of toxicity at the concentration of 0.75㎎/㎖ and at the same concentration the extract caused inhibition of bacterial biofilm formation. The extract of Scrophularia ningpoensis hemsl possesses an in vitro antibacterial antibiofilm activities against K. pneumoniae, with no sign of cytoxicity on L929.

지방산분석을 이용한 Pseudomonas aeruginosa의 동정

육근돌 ( Keun Dol Yook ) 대한임상검사과학회 2005 대한임상검사과학회지(KJCLS) Vol.37 No.3

Cellular fatty acid composition of 17 strains of the Pseudomonas aeruginosa was determined by gas-liquid chromatography isolated from environmental and clinical sample in a C university hospital. Straight-chain saturated acid of C16:0 and straight-chain unsaturated acid of C18:1 with a double bond were commonly found in all the strains tested. The presence of 12:0 3OH (3-10%), 16:0 (18-28%), and 18:1w7c (17-37%) showed the characteristics of the species in the Pseudomonas. Bacterial fatty acid composition was considered to be useful for the study of interrelation and for rapid identification of the bacteria.

Extended-Spectrum β-Lactamase 생성 Klebsiella pneumoniae 균주의 유전형 검출

육근돌(Yook, Keun-Dol),양병선(Yang, Byoung-Seon),박진숙(Park, Jin-Sook) 한국산학기술학회 2013 한국산학기술학회논문지 Vol.14 No.3

임상검체에서 분리되는 그람음성 막대균의 제 3세대 cephalosporin에 대한 내성율의 증가는 임상적으로 심각한 문제가 되고 있다. 3세대 cephalosporin 및 monobactam계 항균제에 대한 내성은 주로 Extended-Spectrum β-Lactamase (ESBL)의 생성에 기인한다. 따라서 ESBL 유전자의 정확한 검출은 병원내의 감염경로 파악을 위한 감시 및 역학조사를 위해 필수적이다. 본 연구는 2012년 2월부터 8월까지 대전, 충남, 충북지역의 대학병원으로부터 ESBL 생성 Klebsiella penumoniae 46균주를 분리하여 Clinical and Laboratory Standards Institute (CLSI)에 따라 ceftazidime (CAZ)과 CAZ/clavulanate (CLA)를 이용한 combination disk test (CDT) 방법에 의해 표현형을 조사하고, 유전형 특이 프라이머를 이용한 multiplex PCR을 수행하여 유전형을 검출하였다. CDT 결과 42균주가 ESBL생성균주로 확인되었다. PCR 결과, 46균주 모두 TEM형이었으며, 37균주는 SHV형, 14균주는 CTX-M형으로 나타났으며 10균주가 TEM, SHV, CTX-M 유전 자를 모두 가지고 있었다. Multiplex PCR에 의한 유전형 검출 방법은 임상에서 분리한 ESBL생성 K. penumoniae균주의 감별과 검출에 유용한 방법으로 사료된다. Among Gram-negative pathogens in Korea, the incidence of resistance to third generation cephalosporins is becoming an ever-increasing problem. The production of extended-spectrum β-lactamase (ESBL) is the main mechanism of bacterial resistance to a third-generation cephalosporins and monobactams. Accurate identification of the ESBL genes are necessary for surveillance and epidemiological studies of the mode of transmission in the hospital. This study was conducted to detect the genes encoding ESBL of 46 K. penumoniae isolated from Daejeon, Chungnam and Chungbuk regional university hospitals from February to August in 2012. The phenotypes of the isolated specimens were examined according to the combination disc test (CDT) by the Clinical and Laboratory Standards Institute (CLSI). Forty two ESBL producing K. penumoniae isolates could be detected using ceftazidime (CAZ) discs with and without clavulanate (CLA). By CDT, 42 K. pneumoniae strains were confirmed to be ESBL strains. Genotyping was performed by multiplex PCR with type-specific primers. By PCR analysis, TEM gene in 46 strains, SHV gene in 37 strains and CTX-M genes in 14 strains were identified. Ten isolates did carry genes encoding ESBLs of all types TEM, SHV and CTX-M. The multiplex polymerase chain reaction (PCR) analysis was better to detect and differentiate ESBL producing K. penumoniae strains in clinical isolates.

대전지역에서 그람음성간균의 항생제 내성률 조사

육근돌 ( Keun Dol Yook ) 대한임상검사과학회 2007 대한임상검사과학회지(KJCLS) Vol.39 No.3

During 2006, the antibiotic resistance rate were investigated in gram negative bacteria. Resistance to piperacillin were detected at 60% in Escherichia coli, and 37% in Klebsiella pneumoniae. Ceftriaxone were detected 58% in Enterobacter cloacae, 52% in Acinetobacter baumannii, 43% in Enterobacter aerogenes and 76% were detected in Serratia marcescens. Between 1998 and 2007 antibiotic resistance rate were decreased in seven types antibiotic drugs. but, ceftazidime were increased from 12 to 20% during this times. In addition, E. coli, E. cloacae, A. baumannii and E. aerogenes were more isolated from May to June and K. pneumoniae and S. marcescens were more isolated from July to September. We should monitor and control antibiotic use and regularly survey antibiotic resistance patterns among pathogens in the hospital.

충청 지역에서 분리된 대장균이 생성하는 Extended Spectrum β-Lactamase 유형의 검출 빈도

육근돌(Yook, Keun-Dol),박진숙(Park, Jin-Sook) 한국산학기술학회 2014 한국산학기술학회논문지 Vol.15 No.4

장내세균 가운데서 가장 빈번하게 나오는 대장균에서 Extended-Spectrum β-Lactamase(ESBL)를 생성하는 효소의 검출빈도와 이들 균주의 ESBL효소의 유형을 구분하는 것이 이 연구의 목적이다.균주는 충청지역 (대전,충남,충북)병원에 서 2013년 2월부터 7월까지 6개월간 282대장균 균주 중 ESBL을 생성하는 74균주(26.2%)를 수집하였다.충청지역에 ESBL 균주를 포함한 대장균의 항균제 내성률은 Aztreonam 30.8%,Cefotaxim 30.9%,그리고 Ceftazidime32.2%로 나타났으며 ESBL을 생성하는 균주는 β-lactam 항균제인 Aztreonam에 58.1%,Cefotaxim 100%,그리고 Ceftazidime63.5%의 내성률을 보였다.동정된 ESBL균의 CTX-M-2은 48균주,CTX-M-8은 20균주,PER-128균주,그리고 VEB-126균주에서 나왔 다.GES-1형은 74균주 중 2균주가 충남에서만 유일하게 나타났다.ESBL검출빈도와 유형의 정확한 파악은 병원감염관리와 항균제 처방에 도움이 될 것이다. The study aims primarily to evaluate the resistance of antibiotics and the prevalence of these enzymes among Escherichia coli the most frequent isolate of Enterobacteriaceae producing Extended-Spectrum β -Lactamase(ESBLs), to differentiate the types of enzymes in these isolates. Total 74(26.2%) Strains of producing ESBLs among the 282 E. coli isolates were isolated from hospitals of Chungcheong area (Daejeon, Chungnam, and Chungbuk) during a 6 month-period from February to July, 2013. 282 E. coli isolates including ESBL shown resistance rates of aztreonam 30.8%, Cefotaxim 30.9%, and Ceftazidime 32.2%, 74 isolates producing ESBLs in E. coli were resistant rates to Aztreonam 58.1%, Cefotaxim 100%, and Ceftazidime 63.5% of β-lactam antibiotics. CTX-M-2 (48 isolates) was the most prevalent type of ESBLs identified. Followed the order of frequency by PER-1 (28 isolates), VEB-1 (26 isolates) and CTX-M-8 (20 isolates), of the 74 isolates, 2 isolates only showed GES-1 in Chungnam province. Accurate identification type of ESBLs would aid in hospital infection control. This would give aid to the physician to prescribe more appropriate antibiotics.

병원성 세균에 대한 마늘추출물의 항균효과

문원희,육근돌 한국디지털정책학회 2014 디지털융복합연구 Vol.12 No.10

SHV ESBL생성 Klebsiella pneumoniae 균주의 실시간중합효소반응분석

양병선,육근돌 대한임상검사과학회 2009 대한임상검사과학회지(KJCLS) Vol.41 No.4

The production of extended-spectrum β-lactamases (ESBLS) of the TEM or SHV type by bacterial pathogens is a major threat to the use of the clinically important expanded-spectrum cephalosporins. The characterization of the SHV ESBLs producing Klebsiella pneumoniae strains present in clinical isolates is time-consuming processes. We describe here in the development of a novel system, which consists of a real time PCR. We found 11 K. pneumoniae strains to be presumptive strains ESBLs producers by clinical and laboratory standards institute (CLSI) guidelines. The double disk synergy test showed 8 ESBL positive andconventional PCR showed 10 SHV ESBL positive, which were K. pneumoniae strains isolates. By real time PCR analysis, SHV gene in 11 of 11 strains were identified. When sequencing analysis was compared with real time PCR, both analysis were presented 99% similarity. In this study, we used a rapid, sensitive, and specific real-time PCR (RT-PCR) method for detection of the assay SHV ESBL producing K. pneumoniae strains in clinical isolates.