• Molecules and Cells
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• v.27 no.2
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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.

• Korean Journal of Microbiology
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• v.32 no.1
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• pp.20-27
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• 1994

An inducible ${\beta}$-lactamase gene (bla) was identified and isolated from the chromosomal DNA of multiple drug resistant strains of Staphylococcus aureus. Determined base sequence of bla and of its flanking region was compared with those of bla genes identified on the staphylococcal plasmids pPC1, pI258, pI1071, and pUB101. Base sequence of 843 base-long structural gene of our bla was same as that of pPCl-, pI258-, and pS1-bla. However, HindIII recognition site Which is found in most of the bla genes at 140 base upstream from the structural gene was moved to the site of 370 base upstream from the structural gene. And one of the two direct repeat sequence found in downstream flanking region of pI1071-bla was deleted in our bla. Amino acid sequence homology analysis of the ORF located around HindIII recognition site reveals that this 80 amino acids-long polypeptide is C-terminus of transposase of Tn4001.

• Pediatric Infection and Vaccine
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• v.22 no.1
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• pp.29-35
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• 2015

Purpose: The purpose of this study was to investigate the clinical characteristics and outcome of febrile urinary tract infections (UTIs) caused by community-acquired extended-spectrum ${\beta}$-lactamase (CA-ESBL)-producing and -nonproducing bacteria. Methods: We analyzed febrile UTIs in children hospitalized at Gachon University Gil Medical Center from January 2011 to December 2013 through retrospective data collection from their medical records. Results: Among pathogens causing 374 episodes of UTIs, the proportion of ESBL-producing bacteria was 13.1% (49/374). The proportion of ESBL-producing Escherichia coli and Klebsiella spp. was 13.6% (48/354) and 5.0% (1/20), respectively. There was no significant difference between the CA-ESBL and CA non-ESBL groups in duration of fever ($4.2{\pm}2.7$ vs. $3.7{\pm}2.1$ days, P=0.10) and bacterial eradication rate with empirical antibiotics (100% vs. 100%). The risk of cortical defects on renal scan significantly depended on existence of vesicoureteral reflux rather than ESBL production of pathogen. Conclusions: There was no significant difference between the CA-ESBL and CA non-ESBL groups in renal cortical defects and clinical outcome. Careful choice of antibiotics is important for treatment of community-acquired UTI in children.

• Korean Journal of Microbiology
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• v.45 no.2
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• pp.163-169
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• 2009

This study was performed to investigate the type of extended-spectrum $\beta$-lactamases (ESBL) produced by bacteria isolated from the sewage of wastewater treatment plant at Minragdong, Suyong-gu in Busan. The facility is located at sushi restaurants and guides its drain water to the wastewater treatment plant at Yonghodong, Nam-gu in Busan. Samples were collected on January, 2009. A total of 19 strains were selected as potential ESBL positive strains through a double disk synergy test. On the basis of the results from biochemical tests including indole, methyl-red, Voges-Proskauer, Simmon's citrate, decarboxylase-dihydrolase and sugar-fermentation tests, the 19 strains were identified with 16 strains of Escherichia coli and 3 strains of Klebsiella pneumoniae. Out of 19 strains, 4 transconjugants against Escherichia coli J53, which is sodium azide resistant recipient strain, were obtained. The plasmids isolated from transconjugants were used for PCR analysis. The type of each extended-spectrum $\beta$-lactamase (ESBL) produced by the strains was determined on the basis of isoelectric focusing analysis and DNA sequencing. The results indicated that the types of ESBL from Klebsiella pneumoniae were SHV-12 (3 strains), and Escherichia coli was SHV-12/TEM-1 (1 strain), respectively.

• Korean Journal of Clinical Laboratory Science
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• v.38 no.1
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• pp.26-33
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• 2006

Recently, the rapid increase in extended-spectrum ${\beta}$-lactamase (ESBL) producing clinical isolates has become a serious problem. In this study, the epidemiologic features and molecular characteristics of ESBL among clinical isolates of Escherichia coli and Klebsiella pneumoniae, antibiotic susceptibility testing, genotype of the ESBL and patterns of chromosomal DNA from PFGE (pulsed field gel electrophoresis) were observed. A total of 53 ESBL-producing clinical isolates (30 of E. coli and 23 of Klebsiella pneumoniae) were collected from two university hospitals in the period of June to July in 2002 and 2003 respectively. The antibiotic resistance frequency of those 53 strains was tested by the disk agar diffusion method with the result that all the strains were resistant to cephalothin. To other antibiotics, the resistance rates of E. coli (30 isolates) were in order of ceftazidime (90.0%), cefotaxime and aztreonam (respectively 83.3%). Also, the resistance rates of K. pneumoniae (23 isolates) were in order of aztreonam (78.3%), ceftazidime (73.9%) and cefotaxime (65.3%). Also the sensitivity of ceftazidime-clavulanic acid were 100% in E. coli and 95.7% in K. pneumoniae. And the sensitivity of cefotaxime-clavulanic acid was 96.7% in E. coli and 91.3% in K. pneumoniae. The types of the ESBL genes were determined by using polymerase chain reaction (PCR). Among the 30 isolates of ESBL-producing E. coli, 6 (20.0%) have SHV only, 5 (16.7%) have TEM only and, 18 (60.0%) have both of TEM and SHV. Among the 23 isolates of ESBL-producing K. pneumoniae, 7 (30.4%) have SHV only, 2 (8.7%) have TEM only, and 14 (60.9%) have both of TEM and SHV. These results show that 52 strains, with only one exception, were confirmed as either TEM or SHV. The patterns of Xba I-digested chromosomal DNA of ESBL-producing E. coli and K. pneumoniae isolates were analyzed by PFGE. PFGE patterns of E. coli and K. pneumoniae were multiclonal, but many strains were grouped into a few types. Therefore, it seems that there were clonal outbreaks or possible horizontal spread. In conclusion, the TEM and SHV ${\beta}$-lactamase are most widely spread in E. coli and K. pneumoniae in Korea. As these types are usually carried by plasmids, the spread of these ${\beta}$-lactamase genes could compromise the future usefulness of third generation cephalosporins for the treatment of infections caused by E. coli and K. pneumoniae.

• Journal of Life Science
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• v.23 no.5
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• pp.703-709
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• 2013

The aim of this study was to investigate the prevalence of Extended-spectrum ${\beta}$-lactamase (ESBL) gene and quinolone resistance determinant (qnr) and the pattern of antibiotic resistance in the ESBL-producing Escherichia coli clinical isolates. The 42 ESBL-producing strains from total 274 isolates were detected using a double disk synergy test. They were isolated from various specimens, such as urine (28 strains), sputum (6 strains), pus (3 strains), wound (2 strains), blood (2 strains), and tissue (1 strain). Using the PCR with the specific primers ESBL, ESBL and qnr gene types were determined. Thirty-five strains possessed one or two ESBL genes. CTX-M-1 type was the most abundant followed by CTX-M-9 type and TEM, but SHV, CTX-M-2, and CTX-M-8 gene types were not detected. qnr gene types were detected from ten isolates in the order of qnrB4, qnrB1, and qnrS. Coexistence of ESBL and qnr genes was found. ESBL-producing isolates showed high resistance against some antibiotics, such as cefotaxmie (80.0%), levofloxacin (82.9%), and ampicillin (100%). Neither a synergy effect from the coexistence of ESBL and qnr genes on antibiotic resistance nor a correlation between the production of qnr gene and quinolone resistance were found.

• Journal of Life Science
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• v.22 no.9
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• pp.1268-1276
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• 2012

The emergence and dissemination of carbapenem-resistant bacteria have resulted in limitations of antibiotic treatment and potential outbreaks of metallo-${\beta}$-lactamase (MBL) producing Pseudomonas aeruginosa resistant to carbapenems. In this study, we conducted molecular characterization of the MBL genes of the ${\beta}$-lactam drug-resistant P. aeruginosa and prepared basic data for treatment and prevention of proliferation of antimicrobial-resistant bacterial infections. Forty-two P. aeruginosa isolates of 254 were resistant to imipenem or meropenem. Among the 42 isolates, 28 isolates were positive for the Hodge test, and 23 isolates were positive for the EDTA-disk synergy test (EDST). MBLs were detected in 59.5% (25/42) of P. aeruginosa isolates. Eight isolates harbored $bla_{IMP-6}$, whereas 17 isolates harbored $bla_{VIM-2}$. The $bla_{IMP-6}$ gene was in a class 1 integron containing five gene cassettes: $bla_{IMP-6}$, qac, aacA4, $bla_{OXA-1}$, and aadA1. Some strains that produce IMP-6 and VIM-2 showed epidemiological relationships. The $bla_{IMP-6}$ gene in carbapenem-resistant P. aeruginosa showed an identical pattern to a gene cassette that was reported at a hospital in Daegu, Korea. Therefore, MBL-producing P. aeruginosa is already endemic in the community. We are concerned that the existence of carbapenem-resistant bacteria containing the blaMBL gene may increase pressure on antibiotic selection when treating infections. We believe that we should select appropriate antibiotics based on the antibiotic susceptibility test and continue the research to prohibit the emergence and spread of antibiotics resistant bacteria.

• Korean Journal of Veterinary Service
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• v.43 no.2
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• pp.79-88
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• 2020

The aim of this study was to investigate the prevalence of CTX-M β-lactamase and plasmid-mediated quinolone resistance (PMQR) genes, and the pattern of antibiotic resistance in cefotaxime-resistant gramnegative bacteria. A total 126 gram-negative bacteria were isolated from hospitalized dogs and cats between 2018 and 2019. The most predominant isolates were E. coli (n=41), followed by Pseudomonas aeruginosa (n=25), Proteus mirabilis (n=14), Klebsiella pneumoniae (n=9), Sphingomonas paucimobilis (n=7), and Enterobacter cloacae and Serratia marcescens (respectively, n=5). Cefotaxime-resistant isolates were identified in 26.2% (33 isolates) of 126 gram-negative bacteria. CTX-M type β-lactamase were found in 15 isolates (10 E. coli, 1 Ent, cloacae and 4 K. pneumoniae, respectively). Among the CTX-M producing gram-negative bacteria, CTX-M-1 and CTX-M-9 were detected in 10 (66.7%) and 5 (33.3%) isolates, respectively. While, CTX-M-2 and CTX-M-8 were not found. PMQR genes were detected in 12 (36.4%) isolates (4 E. coli, 2 Ent, cloacae and 6 K. pneumoniae, respectively), and the predominant PMQR gene was aac(6')-lb-cr (n=9), followed by qnrB (n=8) and qnrS (n=1) alone or in combination. qnrA and qepA were not found. Additionally, 9 (60%) of 12 PMQR positive isolates were co-existence with CTX-M-1 or CTX-M-9. CTX-M or PMQR producing isolates showed highly resistance to penicillins (100%), cephalosporins (100~66.7%), monobactams (72.2%), and non-β-lactam antibiotics (94.4~61.1%) such as quinolones, trimethoprim/sulfamethoxazole, tetracycline and gentamicin. These findings showed CTX-M-1, CTX-M-9, aac(6')-lb-cr and qnrB were highly prevalent in cefotaxime-resistant Enterobacteriaceae isolates from companion animals in our region. Moreover, PMQR genes were closely associated with CTX-M type β-lactamase.

• Korean Journal of Clinical Laboratory Science
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• v.51 no.4
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• pp.444-452
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• 2019

Carbapenem resistance, mediated by the major acquired metallo-β-lactamase (MBL) genes, has been increasingly reported, particularly for clinical isolates of Acinetobacter spp. Of the 191 nonduplicate clinical isolates of the carbapenem-nonsusceptible Acinetobacter spp. evaluated, 125 isolates (65.4%) were positive for the modified imipenem or meropenem-Hodge test, and 49 isolates (25.7%) were positive for the imipenem-EDTA+SMA double disk synergy test (DDS). PCR and sequencing of the bla_VIM-2-allele and bla_IMP-1-allele showed that 29 A. baumannii isolates and 1 A. calcoaceticus isolate had bla_VIM-2, whereas 16 A. baumannii isolates and 2 A. calcoaceticus isolates had bla_IMP-6; 1 isolate of the A. genomospecies 3 had bla_VIM-2 and bla_AIM-1. All the above MBL genes belong to class 1 integron. The size of class 1 integron encompassing bla_VIM-2 or bla_IMP-6 ranges from 2.8 kb to 3.2 kb in clinical isolates of A. baumannii, and 3.2 kb to 3.5 kb in clinical isolates of A. genomospecies 3. bla_VIM-2 was most often located first or second in the class 1 integron, and these integrons often included aacA4. Due to dispersion of the MBL-producing Acinetobacter spp. as well as integron, which may encompass various resistance genes, there is an expectation for the increase of multidrug resistant Gram-negative bacteria, including resistance of carbapenems such as imipenem or meropenem. Hence, the development of new antimicrobial agents for treating severe Acinetobacter spp. infections is needed.

• Journal of the Korean Applied Science and Technology
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• v.39 no.6
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• pp.916-923
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• 2022

Water samples were collected from three spots(Namcheon, Changwoncheon and Cheongwoonji) in Changwon and genomic DNA was isolated from them. Quantitative PCR was performed with the isolated DNA as template and primers targeting five different class A extended-spectrum beta-lactamase(ESBL) genes(bla_OXA-1, bla_SHV, bla_TEM, bla_CTX-M-1, bla_CTX-M-9). The number of total ESBL genes from each sample showed large variations between each sample. Thirty nanograms of DNA from Namcheon contained 1.93×10⁶ copies of ESBL genes whereas the same amount of DNA from Changwoncheon contained 1.47×10⁵ copies of ESBL genes. However, the same amount of DNA from Cheongwoonji pond contained only 9.5×10³ copies of ESBL genes. The ratio of each ESBL genes showed little difference between Namcheon river and Changwoncheon river, but DNA from Cheongwoonji pond showed a large difference from the rest. bla_OXA-1 gene was present at 65.3%, and bla_CTX-M-1 gene 33.6% for Namcheon comprising together almost 99%. bla_OXA-1 gene was present at 64.1%, and bla_CTX-M-1 gene 19.1% for Changwoncheon comprising together over 83%. bla_CTX-M-1 gene was present at 87.5% and bla_CTX-M-9 genes 9.8% for Cheongwoonji, a pond which is a closed and isolated environment.