Seung-Yeol Son

Analysis of a novel class 1 integron containing metallo-β-lactamase gene VIM-2 in Pseudomonas aeruginosa

Carbapenems such as imipenem are stable to most β-lactamases. Recently, increased numbers of carbapenemase producing Gram-negative bacterial strains have been isolated because of the increased use of cabapenems. In this respect, control of these infectious carbapenemase producing Gram-negative bacteria and understanding their resistance mechanism are becoming more important. These carbapenem-hydrolyzing β-lactamase genes have been reported to exist mostly as gene cassettes in an integron. This implies that antibiotic resistance genes may be transferred to other bacteria via the integron. In the present study, we identified and analyzed an integron containing VIM-2 type metallo-β-lactamase gene in a carbapenemase producing Pseudomonas aeruginosa. In addition, the possibility of resistance spread by integron located in a plasmid was tested. Among glucose non-fermenting Gram-negative bacilli with reduced imipenem susceptibility (MIC≥8 μg/ml) isolated from Korean patients, P. aeruginosa 1082 showed resistance to most β-lactams, cephalosporin, and aminoglycoside. We found that P. aeruginosa 1082 was inhibited by EDTA in EDTA double disk synergy test which means that this strain produces metallo-β-lactamase. Class 1 integron containing blaVIM-2 (carbapenem resistance gene), qacF (quaternary ammonium compound resistance gene), aacA4 (aminoglycoside resistance gene), catB3 (chloramphenicol resistance gene), blaoxa-30 (extended-spectrum β-lactam resistance gene), and aadAl (aminoglycoside resistance gene) gene cassettes was detected in P. aeruginosa 1082. The size of the integron was 5,246 bp and the structure and arrangement of the integron was a novel one in comparison with other integrons found in other P. aeruginosa. The integron could be transferred to Escherichia coli JM109 from P. aeruginosa 1082 possibly via self-transferable plasmid DNA. The integron and a blaVIM-2 gene were detected in the plasmid DNA of the transconjugants whose imipenem resistance was slightly increased as a result of accepting the integron from the donor strain.

Human-Specific HERV-K Insertion Causes Genomic Variations in the Human Genome

Human endogenous retroviruses (HERV) sequences account for about 8% of the human genome. Through comparative genomics and literature mining, we identified a total of 29 human-specific HERV-K insertions. We characterized them focusing on their structure and flanking sequence. The results showed that four of the human-specific HERV-K insertions deleted human genomic sequences via non-classical insertion mechanisms. Interestingly, two of the human-specific HERV-K insertion loci contained two HERV-K internals and three LTR elements, a pattern which could be explained by LTR-LTR ectopic recombination or template switching. In addition, we conducted a polymorphic test and observed that twelve out of the 29 elements are polymorphic in the human population. In conclusion, human-specific HERV-K elements have inserted into human genome since the divergence of human and chimpanzee, causing human genomic changes. Thus, we believe that human-specific HERV-K activity has contributed to the genomic divergence between humans and chimpanzees, as well as within the human population.

Human Genomic Deletions Generated by SVA-Associated Events

Mobile elements are responsible for half of the human genome. Among the elements, L1 and Alu are most ubiquitous. They use L1 enzymatic machinery to move in their host genomes. A significant amount of research has been conducted about these two elements. The results showed that these two elements have played important roles in generating genomic variations between human and chimpanzee lineages and even within a species, through various mechanisms. SVA elements are a third type of mobile element which uses the L1 enzymatic machinery to propagate in the human genome but has not been studied much relative to the other elements. Here, we attempt the first identification of the human genomic deletions caused by SVA elements, through the comparison of human and chimpanzee genome sequences. We identified 13 SVA recombination-associated deletions (SRADs) and 13 SVA insertion-mediated deletions (SIMDs) in the human genome and characterized them, focusing on deletion size and the mechanisms causing the events. The results showed that the SRADs and SIMDs have deleted 15,752 and 30,785 bp, respectively, in the human genome since the divergence of human and chimpanzee and that SRADs were caused by two different mechanisms, nonhomologous end joining and nonallelic homologous recombination.

Silver loading on poly(ethylene terephthalate) fabrics using silver carbamate via thermal reduction

Silver-coated poly(ethylene terephthalate) (PET) fabrics were prepared through the deposition of silver nanoparticles (AgNPs) generated from thermal reduction of silver 2-ethylhexylcarbamate. The PET fabrics modified with 3-mercaptopropyltriethoxysilane, which acts as an anchor for the AgNPs, were padded with a solution of silver 2-ethylhexylcarbamate in methanol, and were then reduced for in situ generation of Ag-NPs only by heating at 130 °C. The PET/Ag fabrics were characterized by using scanning electron microscopy (SEM), X-ray diffraction (XRD), energy dispersive X-ray (EDX), X-ray photoelectron spectroscopy (XPS), and water contact angle analysis, and by measuring the electrical conductivity and antibacterial activity. A continuous layer of AgNPs with a size from 30 to 100 nm was assembled on the PET fabrics. The PET/Ag imparts high conductivity to the textiles with an electric resistance as low as 3.44±0.12 Ω, and the antibacterial effects of the treated PET fabric against Escherichia coli O157:H7 (ATCC 43889) and Staphylococcus aureus (ATCC 25923) were examined and were found to be excellent.

Biologically active and C-amidated hinnavinII-38-Asn produced from a Trx fusion construct in Escherichia coli

The cabbage butterfly (Artogeia rapae) antimicrobial peptide hinnavinII as a member of cecropin family is synthesized as 37 residues in size with an amidated lysine at C-terminus and shows the humoral immune response to a bacterial invasion. In this work, a synthetic gene for hinnavinII-38-Asn (HIN) with an additional amino acid asparagine residue containing amide group at C-terminus was cloned into pET-32a(+) vector to allow expression of HIN as a Trx fusion protein in Escherichia coli strain BL21 (DE3) pLysS. The resulting expression level of the fusion protein Trx-HIN could reach 15–20% of the total cell proteins and more than 70% of the target proteins were in soluble form. The fusion protein could be purified successfully by HiTrap Chelating HP column and a high yield of 15 mg purified fusion protein was obtained from 80 ml E. coli culture. Recombinant HIN was readily obtained by enterokinase cleavage of the fusion protein followed by FPLC chromatography, and 3.18 mg pure active recombinant HIN was obtained from 80 ml culture. The molecular mass of recombinant HIN determined by MALDI-TOF mass spectrometer is 4252.084 Da which matches the theoretical mass (4252.0 Da) of HIN. Comparing the antimicrobial activities of the recombinant hinnavinII with C-amidated terminus to that without an amidated C-terminus, we found that the amide of asparagine at C-terminus of hinnavinII improved its potency on certain microorganism such as E. coli, Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus.

Whole-exome sequencing in Tricho-rhino-phalangeal syndrome (TRPS) type I in a Korean family

Tricho-rhino-phalangeal syndrome (TRPS) is a rare autosomal dominant and monogenic disease. Among three types of TRPS, it is known that TRPS type I and type III are caused by deletions or substitutions in the TRPS1 gene, located on chromosome 8 (8q23.3). Although the mutations in TRPS1 gene are responsible for human TRPS, some cases are not detected by the mutations of TRPS1 gene and several cases are presented with different genetic variations. The present case was a sporadic and without TRPS1 mutation. Therefore, we performed whole-exome sequencing (WES) with one patient and his family (father, mother, and brother) and validated novel mutations using PCR and Sanger sequencing. Through family-based WES, we found the two de novo mutations such as ZNF 134 and EXD 3 genes. Through functional effect prediction using disease association Ensembl database, we propose that the de novo mutation of ZNF134 (p.Ser207Arg) could be one of potential candidate genes for causing TRPS and develope the TRPS phenotype in the present case.

Analysis of class I integrons responsible for antibiotics resistance in Pseudomonas aeruginosa

An increase in resistance to antibiotics in Pseudomonasaeruginosa has become a major worldwide concern. Particularly, P. aeruginosa which produces carbapenemases, versatile metallo-β-lactamases (MBLs), causes limitation to treat infection. Class I integrons play an important role in the spread of antibiotic resistance determinants such as MBLs in P. aeruginosa. In the present study, we identified and characterized genetic contents of class I integrons in P. aeruginosa DK45 that was isolated from catheterized urine of patients at Chungbuk National University Hospital in South Korea and strongly exhibited carbapenemase activity. The correlation between integron-associated gene cassettes and antibiotics resistance was also investigated. Screening of the integrons in P. aeruginosa was performed by PCR and the integron-associated gene cassettes were further characterized by DNA sequencing. It was confirmed that three integrons (class I) harboring VIM-2, OXA-10, and OXA-30 type β-lactamase genes, respectively, existed on the bacterial plasmid. Each integron also contained genes encoding resistance determinants to various antibiotics such as aminoglycoside, fluoroquinolone and folate pathway inhibitors. Functional analysis by recombinant plasmids bearing each integron in Escherichiacoli indicated that each integron was associated to a high resistance rate to β-lactam and aminoglycoside antibiotics. The combined results suggest that genetic diversity of class I integrons is a major factor capable of increasing a broad spectrum resistance to the antibiotics.

Comparative study of different molecular methods for typing of Acinetobacter baumannii clinical isolates from University Hospitals

AbstractAcinetobacter baumannii, a rod-shape Gram-negative bacterium, is an opportunistic pathogen causing diseases in humans. This bacterium has been recognized as one of the leading causes of nosocomial infection which occurs in hospital or hospital-like setting. The antibiotic resistance of A. baumannii could result from the heavy use of antibiotics and has been recognized as a threat to human health. However, prevention against the disease caused by A. baumannii is difficult due to variable host susceptibility against their infections. We isolated 53 bacterial strains from four different university hospitals in South Korea and identified 34 out of the 53 isolates as A. baumannii, based on the nucleotide sequence of 16S rRNA and gpi genes. For the subtyping of the clinical isolates, we used enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and multilocus sequencing typing (MLST) and compared the results. The result of ERIC-PCR showed that there are 14 distinct DNA fingerprint patterns in the 34 A. baumannii clinical isolates. For MLST analysis of the isolates, we amplified and sequenced seven housekeeping genes (gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD) from each isolate. Each unique allelic profile at the concatenated nucleotide sequences of the seven genes was assigned as a sequence type (ST) and six different STs (ST92, ST105, ST138, ST169, ST262, and ST357) were detected in the 34 A. baumannii clinical isolates. Among the six STs, ST138 was the most ubiquitous in the A. baumannii clinical isolates. To examine the regional distribution of the isolates, STs were clustered into clonal complexes based on their similarity to a previously registered central genotype and the clustering was verified by network phylogenetic analysis.