돼지에서 분리한 대장균의 항균제 감수성과 테트라사이클린 내성 유전자의 분포

박인달 고신대학교 의과대학 2009 고신대학교 의과대학 학술지 Vol.24 No.2

Background : The high frequency of antibiotic-resistant bacteria against tetracyclines which are most widely used as animal feed additives contribute to the treatment of animals. And these bacteria are able to spread into human and are getting more difficult to treat for bacterial infections. The aim of this work was to investigate susceptibility of antibiotics and to detect tetracycline resistant genes from swine. Methods : Bacteria were collected by rectal swab of swine from livestock farmhouse. The MICs (minimal inhibitory concentration) of penicillin (Pc), ampicillin (Am), tetracycline (Te) and erythromycin (Em) were determined according to the broth microdilution methodology of the Clinical and Laboratory Standards Institute (CLSI). PCR screening was carried out to identify possible tet genes that contributed to tetracycline resistance. Results : All of the 61 isolates were Escherichia coli (E. coli) and were examined for MICs. MIC of antibiotics for the isolates ranged from 32 to ≥256 μg/ml for Pc, 4 to ≥256 μg/ml for Am, 32 to ≥256 μg/ml for Te, and 8 to ≥256 μg/ml for Em. Of the 61 strains analysed by PCR for the presence of the tetracycline resistance genes [tetA, tetB, tetC, tetD, tetE, tetG, tetGG, tetK, tetL, tetM, tetO, tetS, tetA(P), tetQ, and tetX], the most common determinants were tet(A) (61/61, 100%) and tet(B) (13/61, 21.3%). Tet(M) (6/61, 9.8%) were also found. Thirteen strains contained tet(A) and tet(B) genes (21.3%), 4 strains contained tet(A) and tet(M) genes (6.6%), and 2 strains contained tet(A), tet(B), and tet(M) genes (3.3%). Conclusion : All the isolates were highly resistant to Te (MIC, 32 - ≥256 μg/ml) and contained at least 1 of 15 tetracycline resistance genes. However, the presence of more than one resistance determinants did not lead to noticeably higher MICs.

인위적으로 유도한 퀴놀론제제 내성 Mycoplasma hominis의 gyrA, parC, parE 유전자의 돌연변이 분석

박인달 고신대학교의과대학 2007 고신대학교 의과대학 학술지 Vol.22 No.2

Background/Aims : Fluoroquinolones are broad-spectrum antibacterial agents that inhibit DNA gyrase and topoisomerase IV activities. Both enzymes are encoded by the gyrA and gyrB genes for DNA gyrase and parC and parE genes for topoisomerase IV. Mutations in the quinolone resistance-determining regions (QRDRs) of GyrA and ParC mainly and GyrB and ParE less frequently have been described as the major mechanism for quinolone resistance. The aim of the study described here is to investigate the relationship between mutations in the gyrA, parC, and parE Gene and cirofloxacin resistance in M.hominis. Methods : Ciprofloxacin-resistant mutants of M.hominis (Mycoplasma hominis) were generated by stepwise selection at increasing drug concentrations. Sequence analysis of PCR products from the strains were used to examine the quinolone resistance-determining regions of the GyrA, ParC, and ParE proteins. Results : Sixteen strains of M.hominis were isolated and were examined for susceptibility to ciprofloxacin, levofloxacin and moxifloxacin. The MIC range for M.hominis were as follows: ciprofloxacin, 0.5-8 ㎍/㎖; levofloxacin, 0.13-2 ㎍/㎖; moxifloxacin, ≤ 0.06-0.13 ㎍/㎖. M.hominis was highly susceptible to moxifloxacin. Ciprofloxacin - resistant mutants were isolated by serial passing of M.hominis M30 in broth culture. Two mutants, C10-2 and C21, displaying high-level ciprofloxacin resistance (MIC > 128 ㎍/㎖) were found to have a change in GyrA at Ser83 to Leu and in ParC at Ser80 to Ile or Glu84 to Lys, but no changes in ParE. Conclusion : Two mutants displaying high-level ciprofloxacin resistance (MIC > 128 ㎍/㎖) were isolated and the gyrA, parC, and parE genes were sequenced. They harbored amino acid substitution of Ser83 to Leu in the GyrA, and of Ser80 to Ile or Glu84 to Lys in the ParC. These findings indicate that resistance to ciprofloxacin may be due to amino acid substitution in the GyrA and ParC.

닭에서 분리한 장내세균의 테트라사이클린 내성 유전자의 분포

박인달,배일권 고신대학교의과대학 2008 고신대학교 의과대학 학술지 Vol.23 No.4

Background : Widespread resistance to the broad-spectrum tetracyclines has been caused by heavy clinical use and misuse in the human population. Additional contributing factors are the use of tetracyclines as a growth promotor in agriculture and as infection control agent in domestic animals and aquaculture. The purpose of this study was to investigate susceptibility of tetracycline and to detect tetracycline resistant genes from chickens. Methods : Thirty-seven tetracycline resistant enteric bacteria were collected from the rectal swab of chickens by disk diffusion method, and the MICs (minimal inhibitory concentration) of tetracycline, oxytetracycline, chlortetracycline and erythromycin were determined by agar dilution method. PCR screening was carried out to identify possible tet genes that contributed to tetracycline resistance. Results : Of the 37 tetracycline-resistant isolates, the frequency of isolation was Escherichia coli 32 (84.2%), Citrobacter freundii 2 (5.3%), Klebsiella pneumoniae 1 (2.5%), Lectercia adecarboxylata 1 (2.5%), Proteus vulgaris 1 (2.5%). The MIC range for the tetracycline-resistant enteric bacteria was as follows: tetracycline, >256 ㎍/㎖; oxytetracycline, >256 ㎍/㎖; chlortetracycline, 16∼>128 ㎍/㎖; erythromycin, 32∼>256 ㎍/㎖. All of the 37 tetracycline-resistant isolates were positive for tet(A) gene (100%), and 8 (21.6%) of these isolates were found to harbor the tet(A) plus tet(B) genes. Conclusion : All isolates were resistant to tetracycline, oxytetracycline, chlortetracycline and erythromycin, and they harbored tet(A) gene or tet(A) plus tet(B) genes that gave rise to the tetracycline resistance. These results show that enteric bacteria isolates from chicken in Korea are resistant to tetracycline and all of them harbored tet(A) gene.

인위적으로 유도된 목시플로사신 내성 Mycoplasma hominis의 표현형과 유전자형의 연관성

박인달(Indal Park),최명원(Myungwon Choi) 한국생명과학회 2010 생명과학회지 Vol.20 No.10

본 연구에서는 QRDRs의 유전자 돌연변이와 목시플로사신의 농도와의 관계를 알아보기 위하여 목시플로사신의 농도를 단계적으로 높여가며 Mycoplasma hominis (M. hominis)에 작용시켜 목시플로사신에 내성을 갖는 균주 6주(M1, M4, M8, M16, M32, M64)를 만들었고, 이 돌연변이주들의 MIC는 각각 0.5, 4, 8, 16, 32, 64 ㎍/㎖이었다. 이 균들의 염기서열을 분석하였더니 모든 돌연변이주들에서 Arg163Thr (GyrA), Pro445Gln (ParE) 아미노산 치환이 관찰 되었고, 목시플로사신의 농도가 높아질수록 Ser153Lys (GyrA, ≥4 ㎍/㎖), Ser91Ile (ParC, ≥16 ㎍/㎖), Val450Phe (GyrB, ≥64 ㎍/㎖) 등과 같은 아미노산의 치환이 추가로 관찰되었다. 이러한 아미노산의 치환이 목시플로사신의 내성과 연관이 있는 것으로 생각되며, 특히 GyrB 단백질의 아미노산 치환은 목시플로사신의 고도 내성과 연관이 있는 것으로 생각된다. Moxifloxacin (MF) - resistant mutants of Mycoplasma hominis (M. hominis) were generated by stepwise selection in increasing concentrations of MF, and six strains of MF resistant M. hominis mutants - M1, M4, M8, M16, M32, and M64 - in which MICs of MF were 0.5, 4, 8, 16, 32, 64 ㎍/㎖, respectively, were generated. Compared to the sequence of M. hominis PG21, all mutants harbored amino acid substitutions of Arg-163 Thr in GyrA, and Pro-445 Gln in ParE. While the concentrations were getting higher, an additional amino acid substitution was found at Ser-153 Lys in GyrA (≥4 ㎍/㎖), Ser-91 Ile in ParC (≥16 ㎍/㎖), and Val-450 Phe (≥64 ㎍/㎖) in GyrB. These substitutions seem to have an impact on resistance to MF, and GyrB change was found only in the highest concentration and seems to be associated with high-level resistance to MF. This, as far as we know, is the first description of a relationship between MF resistance phenotype and genotype.

Tetracycline 저항성 세균에서 tetM유전자의 분포

박인달(In-Dal Park),김광혁(Kwang-Hyuk Kim),장명웅(Myung-Woong Chang),박형숙(Hyung-Sook Park) 大韓微生物學會 1992 大韓微生物學會誌 Vol.27 No.1

Mycoplasma Hominis에서 분리한 Type II-DNA Topoisomerase의 효소반응 특성

정인철,박인달 고신대학교의과대학 2008 고신대학교 의과대학 학술지 Vol.23 No.4

Background : DNA topoisomerases are essential enzymes that catalyze the mutual conversion of the topological states of DNA. Two major classes of enzyme have been isolated from a number of organisms. Nevertheless, the enzymatic properties of DNA topoisomerases in the mycoplasma species has not been identified yet. Methods : We have examined the activities of the DNA topoisomerases from various types of mycoplasma and the characterizations of partially purified type II-topoisomerase fractions from Mycoplasma hominis. Results : The levels of the DNA topoisomerase activities in Mycoplasma hominis and Mycoplasma fermentans were higher than those in Ureaplasma urealyticum, Mycoplasma penetrans and Mycoplasma pneumoniae. DNA topoisomerase has been partially purified from Mycoplasma hominis by Ultrogel AcA34 gel filtration. This enzyme was highly stimulated in relaxation of supercoiled DNA by ATP-Mg2+. Topoisomerase mediated DNA cleavage was enhanced by mAMSA (eukaryotic type II inhibitor) and norfloxacin (prokaryotic type II inhibitor) at various concentrations (0.05-0.5 mM). Conclusion : Our results suggest that enzymatic properties of type II-topoisomerase in Mycoplasma hominis have analogous to type II-human DNA topoisomerase and type IIA-bacterial topoisomerase.

β-lapachone이 염증성 사이토카인 생성에 미치는 효과

최명원,박인달,박건영,김광혁 대한암예방학회 2011 Journal of cancer prevention Vol.16 No.2

β-Lapachone (1,2-naphthoquinone; β-lap) was originally isolated from the bark of the Lapacho tree,and its derivatives have been shown to exhibit a number of pharmacologic actions, including antiviral,antiparasitic, and antitumor activities. In the present study, we investigated the effects of β-lap on the production of cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interferon-γ(IFN-γ) in mice. The culture supernatants of mice splenocytes exposed with cell stimulants (LPS, Con A) alone or β-lap plus lipopolysaccharide (LPS), concanavalin A (Con A) were subjected to assay TNF-α, IL-6, and IFN-γ. TNF-α and IL-6 levels were lower and IFN-γ levels were higher in the culture supernatants of mice splenocytes exposed to Con A+β-lap than those of the groups exposed to Con A alone. These findings demonstrate that β-lap downregulates the inflammatory cytokines immune responses by TNF-α, IL-6 and upregulates anti-inflammatory cytokine by IFN-γ in allergy-associated immunity. These results suggest that β-lap may have a potential for preventive or adjunct antiinflammatory therapy by modulating the production of cytokines. However, further in vivo investigation of this compound's activity is necessary to elaborate its mechanisms. (Cancer Prev Res 16, 155-160,2011)

호흡기질환 환자로부터 분리된 Mycoplasma pneumoniae의 tetracycline과 erythromycin에 대한 저항성 변이

장명웅,박인달,김광혁,송갑영,김성원,Chang Myung-Woong,Park In-Dal,Kim Kwang-Hyuk,Song Gap-Young,Kim Sung-Won 한국생명과학회 2005 생명과학회지 Vol.15 No.6

2002년 2월부터 2005년 4월까지 호흡기질환 환자로부터 분리된 M. pneumeniae 123 균주의 tetracycline과 erythromycin에 대한 MIC 범위는 각각 $0.5\~1.0$, and $0.5\~512{\mu}/ml$ 이었다. 분리된 M. pneumoniae 123 균주에서 plasmid DNA는 확인되지 않았다. 분리된 M. pneumoniae 123 균주 중에서 57($46.3\%$) 균주가 tetracycline에 저항성인 tetM유전자를 가지고 있었으며, 235 rRNA domain V에 erythromycin에 저항성 변이를 일으킨 균주가 60($48.8\%$)이었다. erythromycin에 저항성 변이를 일으키지 않은 63균주 중에서 tetM 유전자를 가지고 있는 균주는 36($57.1\%$)이었으며, erythromycin에 저항성 변이를 일으킨 60균주 중에서 21($35.0\%$ 균주가 tetM 유전자를 가지고 있었다. 본 연구로써 국내에서 tetracycline과 erythromycin에 대한 저항성 M. pneumoniae 균주의 분리율이 외국에 비하여 높으며, M. pneumoniae 감염의 치료에 erythromycin이 일차 선택제가 될 수 없으므로 이에 대한 범국가적 조사가 필요하다고 생각된다. One hundred and twenty three strains of Mycoplasma pneumoniae were isolated from patients with respiratory diseases from February 2002 to April 2005 in Busan, Korea. The MICs of tetracycline and erythromycin up to $90\%$ of the 123 M. pneumoniae isolates tested were $0.5\~1.0$, and $0.5\~512{\mu}/ml$, respectively. Plasmid DNA was not isolated from all of the M. pneumoniae isolates. Out of 323 strains of M. pneumoniae, 57 ($46.3\%$) stains contain tetM gene on their chromosomal DNA, and 60 ($48.8\%$) strains were mutated in domain V of 23S rRNA for erythromycin resistance. Out of 63 strains of M. pneumoniae which were not mutated in domain V of 235 rRNA for erythromycin resistance, 36 ($57.1\%$) strains contained tetM gene, and out of 60 strains of M. pneumoniae which were mutated in domain V of 23S rRNA for erythromycin resistance, 21 ($35.0\%$) strains contained tetM gene. These results suggest that the isolation rate of erythromycin and tetracycline resistant M. pneumoniae is higher than those of other countries, and erythromycin and tetracycline are not first choice drug for M. pneumoniae infection in Korea, and it need confirm by a nationwide surveilance of antimicrobial resistance.

Genetic Classification and Antimicrobial Resistance of Ureaplasma urealyticum Isolated from Urine

최명원,박인달 대한미생물학회 2012 Journal of Bacteriology and Virology Vol.42 No.2

Recently, polymerase chain reaction (PCR)-based methods have been used to reclassify Ureaplasma urealyticum into two independent species (spp.), designating U. parvum and U. urealyticum. In the current study, we aim to reclassify U. urealyticum and to analyze the correlation between the presence of a genetic marker and an antibiotic resistance of U. urealyticum. Susceptibility test against tetracycline, levofloxain or moxifloxacin was performed by broth microdilution method. The presence of tet(M) gene and the mutations of quinolone resistance-determining regions (QRDRs) were analyzed by PCR and sequencing. Among fourteen Ureaplasma isolates, three were identified as U. parvum and eleven were identified as U. urealyticum, and this is first report showing that two independent spp. of U. urealyticum isolated from Korean are present. The minimum inhibitory concentration (MIC) ranges for Ureaplasma isolates were as follows:tetracycline 0.25~128 μg/ml, levofloxacin 1~8 μg/ml, and moxifloxacin 0.5~4 μg/ml. The tet(M) determinant was found in 3 among 14 Ureaplasma isolates with tetracycline MIC of >16 μg/ml, suggesting that the presence of the tet(M) determinant is associated with tetracycline resistance. Mutations of gyrA, gyrB, parC, and parE genes in the QRDRs were found in 3 among 14 Ureaplasma isolates, exhibiting only parE gene mutation is associated with fluoroquinolone resistance.

정상 및 암환자의 자연살해세표(Natural Killer Cell)가 종양표적세포의 성장저해에 미치는 효과

김광혁,박인달,장명웅,구자영,박은동 고신대학교 의과대학 1995 고신대학교의과대학부설연구소논문집 Vol.1 No.-