• Molecules and Cells
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• 제27권2호
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• pp.225-235
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• 2009

Antibody phage display provides a powerful and efficient tool for the discovery and development of monoclonal antibodies for therapeutic and other applications. Antibody clones from synthetic libraries with optimized design features have several distinct advantages that include high stability, high levels of expression, and ease of downstream optimization and engineering. In this study, a fully synthetic human scFv library with six diversified CDRs was constructed by polymerase chain reaction assembly of overlapping oligonucleotides. In order to maximize the functional diversity of the library, a ${\beta}$-lactamase selection strategy was employed in which the assembled scFv gene repertoire was fused to the 5'-end of the ${\beta}$-lactamase gene, and in-frame scFv clones were enriched by carbenicillin selection. A final library with an estimated total diversity of $7.6{\times}10^9$, greater than 70% functional diversity, and diversification of all six CDRs was obtained after insertion of fully randomized CDR-H3 sequences into this proofread repertoire. The performance of the library was validated using a number of target antigens, against which multiple unique scFv sequences with dissociation constants in the nanomolar range were isolated.

• Journal of Microbiology and Biotechnology
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• 제2권2호
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• pp.129-134
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• 1992

Segregational instability of a recombinant plasmid, pDML6, encoding extracellular $\beta$-lactamase in Streptomyces lividans PD6 was characterized by growth kinetic analysis. The quantitative determination of the plasmid harbored in the mycelia was evaluated with mycelia fragmented mechanically, and also with colonies regenerated from protoplasts. Conditions for the formation of protoplasts and regeneration of protoplasts were established. The maximal specific growth rates of the host strain and the plasmid-harboring strain in a chemically defined medium without selection pressure were the same. The probability of plasmid loss from the harbouring cells was higher at higher growth rates. Mathematical models for the prediction of cell growth, substrate uptake, and accumulation of the cloned gene product were developed.

• 대한임상검사과학회지
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• 제38권3호
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• pp.166-172
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• 2006

Metallo-${\beta}$-lactamase (MBL) can hydrolyze all ${\beta}$-lactams except monobactams and frequently coexists with various antibiotic resistance genes such as aminoglycoside resistance, sulfonamide resistance gene, etc. Therefore, the effective antibiotics against infections by these bacteria are markedly limited or can't even be found. We tried to search in-vitro antimicrobial combinations with synergistic effects for a VIM-2 type MBL producing Pseudomonas aeruginosa, isolated from clinical specimen. On the selection of antibiotic combinations with synergistic effects, we performed a one disk synergy test, modified Pestel's method, in agar without aztreonam (AZT). The bacteriostatic synergistic effects of this tests were scored as $S_1$ (by susceptibility pattern in agar without antibiotics), $S_2$ (by the change of susceptibility in agar with or without antibiotics) and $S_3$ ($S_1$ + $S_2$) and was classified into weak (1 point), moderate (2 points) and strong (3 points) by $S_3$ score. Subsequently, we carried out the time-killing curve for the antibiotic combinations with the strong synergistic bacteriostatic effect. One VIM-2 type MBL producing P. aeruginosa confirmed by the PCR showed all resistance against all ${\beta}$-lactams except AZT, aminoglycoside and ciprofloxacin. In the one disk synergy test, this isolate showed a strong bacteriostatic synergistic effect for the antibiotic combination of AZT and piperacillin-tazobactam (PIP-TZP) or AZT and amikacin (AN). On the time-killing curve after six hours of incubation, the colony forming units (CFUs/mL) of this bacteria in the medium broth with both combination antibiotics were decreased to 1/18.7, 1/17.1 of the least CFUs of each single antibiotics. The triple antibiotic combination therapy including AZT, PIP-TZP and AN was shown to be significantly synergistic after 8 hrs of exposure. In a VIM-2 MBL producing P. aeruginosa with susceptibility for AZT, the triple antibiotic combination therapy including AZT, PIP-TZP and AN may be considered as an alternative antibiotics modality against the infection by some MBL type. But the antimicrobial combination therapy for many more MBL producing isolates is essential to know as soon as possible for the selection of effective treatment against the infection by this bacteria.

• 생명과학회지
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• 제22권9호
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• pp.1268-1276
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• 2012

사람의 감염증 치료에 사용되는 carbapenem계 약제에 대한 내성균의 출현 및 확산은 감염증 치료를 제한할 뿐만 아니라 집단 발병의 원인이 될 수 있다. 이에 본 연구에서는 ${\beta}$-lactam 약제에 내성을 갖는 Pseudomonas aeruginosa (P. aeruginosa)를 대상으로 metallo-beta-lactamase (MBL)의 유전형을 규명함으로써 내성세균의 감염증 치료지침 및 확산방지책 마련에 기초 자료를 제공하고자 하였다. 본 연구의 대상이 된 254개의 임상 검체 중에서 42주의 P. aeruginosa 를 분리하여 imipenem 혹은 meropenem에 내성을 나타내는 Hodge 변법과 EDST에서 각각 28주와 23주가 양성반응을 보였다. DNA의 염기서열 분석결과 $bla_{IMP-6}$ 유전자 보유균이 8주, $bla_{VIM-2}$ 유전자 보유균이 17주로 59.5%(25/42)가 MBL을 생성하는 것으로 나타났다. $bla_{IMP-6}$의 유전자 환경은 $bla_{IMP-6}$-qac-aacA4-$bla_{OXA-1}$-aadA1 유전자 배열을 지니고 있었다. 또한 ERIC PCR 결과 IMP-6과 VIM-2를 생성하는 일부 균주에서 역학적 연관성이 있음이 확인되었다. 본 연구에서 분리한 carbapenem계 항균제 내성 P. aeruginosa가 보유한 $bla_{IMP-6}$ 유전자는 대구지역에서 발병이 보고된 유전자의 gene cassette와 일치하는 것으로 확인되었다. 따라서 이들 세균이 지역사회에 정착하고 있고 이들을 보유한 세균에 의한 감염증 치료시 치료약제에 대한 선택압을 증가시킬 것으로 우려된다. 그러므로 항균제 내성 검사를 통하여 적절한 항균제를 선택하고, 항균제 내성균들의 출현과 확산을 막는 연구가 계속되어야 할 것으로 생각된다.

• Journal of Microbiology and Biotechnology
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• 제17권10호
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• pp.1607-1615
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• 2007

We investigated the applicability of the TEM-l ${\beta}$-lactamase fragment complementation (BFC) system to develop a strategy for the screening of protein-protein interactions in bacteria. A BFC system containing a human Fas-associated death domain (hFADD) and human Fas death domain (hFasDD) was generated. The hFADD-hFasDD interaction was verified by cell survivability in ampicillin-containing medium and the colorimetric change of nitrocefin. It was also confirmed by His pull-down assay using cell lysates obtained in selection steps. A coiled-coil helix coiled-coil domain-containing protein 5 (CHCH5) was identified as an interacting protein of human uracil DNA glycosylase (hUNG) from the bacterial BFC cDNA library strategy. The interaction between hUNG and CHCH5 was further confirmed with immunoprecipitation using a mammalian expression system. CHCH5 enhanced the DNA glycosylase activity of hUNG to remove uracil from DNA duplexes containing a U/G mismatch pair. These results suggest that the bacterial BFC cDNA library strategy can be effectively used to identify interacting protein pairs.

• Childhood Kidney Diseases
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• 제8권2호
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• pp.214-222
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• 2004

목적: 소아 요로감염은 적절한 항생제 치료가 중요하며 항생제의 선택에는 E. coli에 대한 항생제 감수성이 기준이 된다. Extended-spectrum ${\beta}-lactamase(ESBL)$은 E. coli 등 그람음성균에서 분비되어 광범위 항생제 내성을 초래하는 효소로서 주로 병원 감염에서 발생하여 치료를 어렵게 하는 요인으로 알려져 있다 저자들은 지역사회 획득 소아 요로감염에서도 ESBL(+) E. coli가 분리되었기에 ESBL(+) E. coli 요로감염의 특성을 후향적으로 조사하고자 하였다. 방법: 2001년 3월부터 2003년 2월까지 이대목동병원 소아과에 입원한 지역사회 획득 E. coli 요로감염 소아(288명)를 대상으로 ESBL을 검사하였다. 검사 방법은 미량 액체배지 희석법을 이용한 자동화 기계(Vitek GNS 433 card)에서 ESBL이 의심되는 균주에 한해 National Committee for Clinical Laboratory Standard (NCCLS)의 기준에 따라 ESBL을 확인하였다. ESBL의 결과에 따라 ESBL(+) E. coli 요로감염의 발생률을 조사하였고 임상적 특징, 위험요인, 항생제 내성률 및 치료효과 등을 ESBL(-) E. coli 요로감염과 비교 분석하였다. 결과:지역사회 획득 소아 요로감염에서 ESBL(+) E. coli의 발생률은 10.8%(31명)이였고 93.5%(29/31)가 6개월 미만의 영아에서 발생하였다(P<0.01). 임상소견은 ESBL 양성군과 음성군 사이에 유의한 차이가 없었다. ESBL 밭생의 위험인자로 잘 알려진 항생제 사용력, 입원병력 및 요로계 기형과도 무관하였다. ESBL(+) E. coli의 항생제 내성률은 ESBL(-) E. coli 에 비하여 유의하게 높았다(P<0.05). Ceftriaxone 투여 48시간 후의 멸균률은 ceftriaxone에 대한 높은 내성률에도 불구하고 96.8%(30/31)로 높았다. 그러나 6개월 이내의 재발률이 25.8%(9/31)나 되었고 이는 ESBL 음성군의 재발률 6.6%(17/227)에 비하여 유의하게 높았다(P<0.05). 결론: 지역사회 획득 소아 요로감염에서 6개월 미만의 어린 영아는 ESBL(+) E. coli 발생의 새로운 위험요인이므로 이에 대한 역학적 연구가 요구된다. 소아 요로감염에서 경험적으로 사용하는 항생제의 선택에 대한 변화가 필요할 것으로 생각된다.

• 고신대학교 의과대학 학술지
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• 제33권2호
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• pp.159-170
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• 2018

Objectives: The purpose of this study was to compare antibiotic resistance patterns between first urinary tract infection (UTI) and recurrent UTI groups and to obtain information regarding empirical antibiotic selection for treating recurrent UTI. Methods: We retrospectively reviewed 148 children treated for UTIs from January 2009 to June 2016. The patients were divided into two groups: first UTI (N = 148) and recurrent UTI (17 patients and 20 episodes). Results: In both groups, Escherichia coli was the most frequent causative organism, accounting for 89.9% and 75.0% in the first and recurrent UTI groups, respectively. When E. coli or Klebsiella pneumoniae was the causative organism, extended-spectrum ${\beta}-lactamase$ (ESBL)-producing organisms were more frequent in the recurrent UTI group (17.6%) than in the first UTI group (14.0%); however, this difference was not statistically significant (P = 0.684). Cefotaxime was the most frequently used first-line empirical antibiotic in both groups. In the first UTI and recurrent UTI groups, 7.4% and 15.0% of patients were treated with intravenous antibiotics as definitive therapy, respectively (P = 0.250). Fifteen out of 17 patients having a second UTI had different causative organisms or antibiotic susceptibility patterns compared to their previous episode. Conclusions: Escherichia coli was the most frequent causative organism in the recurrent UTI group. There were no differences in the proportion of ESBL-producing organisms between the first UTI and recurrent UTI groups. Therefore, when a UTI recurs in children, the antibiotics effective on the most common causative organism might be administered as empirical antibiotics.

• 한국미생물·생명공학회지
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• 제25권5호
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• pp.464-467
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• 1997

A shuttle vector, pSHvec, was constructed for Lactobacillus casei (L. casei) YIT 9018 and JM1O9 by recombinant DNA technology. This vector contained the $\beta$-lactamase II gene from Bacillus cereus as a selection marker and replication origins for both Gram(+) and Gram(-) strains. It could transform the wild type L. casei YIT 9018 as well as E. coli JM109 and transformed cells were selected based on antibiotics resistance. The ability of L. casei YIT 9018 for curd formation in 11% skim milk was maintained even after transformation with pSHvec. The vector was stable as long as antibiotics were added to the medium. These results could contribute to the study of lactic acid bacteria for the industrial purpose at a genetic level.

• 대한임상검사과학회지
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• 제43권2호
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• pp.68-74
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• 2011

Pseudomonas aeruginosa is an aerobic, Gram-negative, glucose-nonfermenting bacterium, which has emerged as a serious opportunistic pathogen. Recently, outbreaks of carbapenem resistant P. aeruginosa give rise to significant therapeutic challenges for treating nosocomial infections. The genes of metallo-${\beta}$-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 the prevalence of integron in imipenem resistant P. aeruginosa isolates. A total of 61 consecutive, non-duplicate, and imipenem resistant P. aeruginosa strains were isolated from a university hospital in the Chungcheong province of Korea. We employed repetitive extragenic palindromic sequence-based PCR (rep-PCR) method for the selection of clonally different P. aerusinosa strains. PCR and DNA sequencing were conducted for the detection of integrons. Twenty-one clonally different P. aeruginosa strains were isolated. Only one (P28) of the strains harbored $bla_{VIM-2}$ that was found as gene cassettes in class 1 integrons. Four of 21 carbapenem resistant P. aeruginosa strains harbored class 1 integron containing aminoglycoside resistance determinant. All of the integrons detected in the study contained more than one resistance gene cassette, which can mediate resistance to multiple antibiotics. To prevent further spreading of the multi-drug resistant P. aeruginosa, conseguent monitoring and clinical polices are required.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2000년도 Proceedings of 2000 KSAM International Symposium and Spring Meeting
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• pp.3-6
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• 2000

Antimicrobial resistance has been a well-recognized problem ever since the introduction of penicillin into clinical use. History of antimicrobial development can be categorized based on the major antibiotics that had been developed against emerging resistant $pathogens^1$. In the first period from 1940 to 1960, penicillin was a dominating antibiotic called as a "magic bullet", although S.aureus armed with penicillinase led antimicrobial era to the second period in 1960s and 1970s. The second stage was characterized by broad-spectrum penicillins and early generation cephalosporins. During this period, nosocomial infections due to gram-negative bacilli became more prevalent, while those caused by S.aureus declined. A variety of new antimicrobial agents with distinct mechanism of action including new generation cephalosporins, monobactams, carbapenems, ${\beta}$-lactamase inhibitors, and quinolones characterized the third period from 1980s to 1990s. However, extensive use of wide variety of antibiotics in the community and hospitals has fueled the crisis in emerging antimicrobial resistance. Newly appeared drug-resistant Streptococcus pneumoniae (DRSP), vancomycin-resistant enterococci (VRE), extended-spectrum ${\beta}$-lactamase-producing Klebsiella, and VRSA have posed a serious threat in many parts of the world. Given the recent epidemiology of antimicrobial resistance and its clinical impact, there is no greater challenge related to emerging infections than the emergence of antibiotic resistance. Problems of antimicrobial resistance can be amplified by the fact that resistant clones or genes can spread within or between the species as well as to geographically distant areas which leads to a global concern$^2$. Antimicrobial resistance is primarily generated and promoted by increased use of antimicrobial agents. Unfortunately, as many as 50 % of prescriptions for antibiotics are reported to be inappropriate$^3$. Injudicious use of antibiotics even for viral upper respiratory infections is a universal phenomenon in every part of the world. The use of large quantities of antibiotics in the animal health industry and farming is another major factor contributing to selection of antibiotic resistance. In addition to these background factors, the tremendous increase in the immunocompromised hosts, popular use of invasive medical interventions, and increase in travel and mixing of human populations are contributing to the resurgence and spread of antimicrobial resistance$^4$. Antimicrobial resistance has critical impact on modem medicine both in clinical and economic aspect. Patients with previously treatable infections may have fatal outcome due to therapeutic failure that is unusual event no more. The potential economic impact of antimicrobial resistance is actually uncountable. With the increase in the problems of resistant organisms in the 21st century, however, additional health care costs for this problem must be enormously increasing.