Analysis of Integrons and Antimicrobial Resistances of Multidrug Resistant Escherichia coli Isolated in Korea

Yun-Yi Yang,서민호 대한미생물학회 2019 Journal of Bacteriology and Virology Vol.49 No.4

Antibacterial drugs are one of the most important therapeutic agents of bacterial infections but multidrug resistant Escherichia coli (MDREC) is an increasing problem worldwide. Major resistance mechanism of MDREC is horizontal gene transfer of R plasmids harboring integrons, which the integron integrase (IntI) catalyzes gene cassette insertion and excision through site specific recombination. In this study, resistance profiles of integron harboring E. coli isolated in Korea and the genetic environments of integron gene cassettes were analyzed by PCR and direct sequencing to clarify the mechanisms of spread of integron harboring E. coli. Resistance rates of integron harboring E. coli, including β-lactams, aminoglycosides, and fluoroquinolones and MDR frequencies were significantly higher than that of E. coli without integron (p<0.01). Majority (80%) of integron harboring E. coli showed resistance transfer by conjugation. Most (80%) of E. coli had dfrA17-aadA5 cassette array and PcH1 hybrid promoter; 16.7% of E. coli had dfrA12-orfF-aadA2 cassette array and PcW promoter. The higher prevalence of weak Pc variants among most (96.7%) of integron harboring MDREC suggests that a flexible cassette array is more important than enhanced expression. All the integrons had LexA binding motif suggests that SOS responses control the expression of these integrons. In conclusion, the genetic bases of integrons were diverse, and the spread and the expression of prevalent gene cassette arrays may be deeply related with strengths of Pc promoters in integrons. These informations will provide important knowledge to control the increase of integron harboring MDREC.

임상 검체에서 분리된 nontyphoid Salmonella에서의 integron의 빈도와 특징

이정은,박수진,김미나,이남용,이복권,이상오,김양수,우준희,최상호,김성한 대한감염학회 2009 Infection and Chemotherapy Vol.41 No.3

Background:Nontyphoid Salmonella (NTS) is a leading cause of human food-borne enteritis. It has been known that integron, a naturally occurring gene capture and expression element, plays an important role in the development and dissemination of multidrug-resistance. In this study, we investigated the prevalences and molecular characteristics of integrons in NTS clinical strains. Materials and Methods:Between 1995-96 and 2000-03, a total 261 NTS clinical strains comprising 39 serotypes were collected from clinical specimens. All strains were serotyped, and the MICs of ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim were determined by agar dilution method. Integrons were detected by PCR amplification of integrase genes, and gene cassettes were determined by PCR and sequencing. Conjugation experiments were performed using E. coli J53 as a recipient. The clonal relationship was analyzed by pulsed-field gel electrophoresis (PFGE). Results:Of the 261 strains tested, class 1 integrons were present in 21 strains (8.0%). Class 2 and class 3 integrons were not found. The integron-positive rate was higher in S. Typhimurium (24.2% [8/33]) than in S. Enteritidis (2.0% [3/153]). Overall rates of antimicrobial resistance were higher in integron-positive strains. dhfr12-orfF-aadA2 gene cassette was detected in 5 strains, aadA2 in 4 strains, dhfr17-orfF-aadA5 in 2 strains, and addA1 in 1 strain. Ten integron-positive transconjugants were successfully selected. Among 8 integron- positive strains of S. Typhimurium, 7 had similar PFGE patterns. Conclusion:This study suggests that integrons are already playing a significant role in antimicrobial resistance in NTS. Continuous monitoring is needed to detect the emergence and spread of integron- mediated antimicrobial resistance. Background:Nontyphoid Salmonella (NTS) is a leading cause of human food-borne enteritis. It has been known that integron, a naturally occurring gene capture and expression element, plays an important role in the development and dissemination of multidrug-resistance. In this study, we investigated the prevalences and molecular characteristics of integrons in NTS clinical strains. Materials and Methods:Between 1995-96 and 2000-03, a total 261 NTS clinical strains comprising 39 serotypes were collected from clinical specimens. All strains were serotyped, and the MICs of ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim were determined by agar dilution method. Integrons were detected by PCR amplification of integrase genes, and gene cassettes were determined by PCR and sequencing. Conjugation experiments were performed using E. coli J53 as a recipient. The clonal relationship was analyzed by pulsed-field gel electrophoresis (PFGE). Results:Of the 261 strains tested, class 1 integrons were present in 21 strains (8.0%). Class 2 and class 3 integrons were not found. The integron-positive rate was higher in S. Typhimurium (24.2% [8/33]) than in S. Enteritidis (2.0% [3/153]). Overall rates of antimicrobial resistance were higher in integron-positive strains. dhfr12-orfF-aadA2 gene cassette was detected in 5 strains, aadA2 in 4 strains, dhfr17-orfF-aadA5 in 2 strains, and addA1 in 1 strain. Ten integron-positive transconjugants were successfully selected. Among 8 integron- positive strains of S. Typhimurium, 7 had similar PFGE patterns. Conclusion:This study suggests that integrons are already playing a significant role in antimicrobial resistance in NTS. Continuous monitoring is needed to detect the emergence and spread of integron- mediated antimicrobial resistance.

경북지역에서 분리된 Salmonella enterica Serovar Typhimurium의 Class I Integron Gene Cassette 특성과 PEGE 유형분석

손창규,이정아,이도영,허완,정중교,Sohn, Chang-Kyu,Lee, Jung-A,Lee, Do-Young,Hun, Wan,Jung, Jung-Kyo 한국미생물학회 2006 미생물학회지 Vol.42 No.1

2003년부터 2004년까지 정북지역에서 발생한 설사환자로부터 17주, 돼지로부터 18주 등 총 35주의 Salmonella enterica serovar Typhimurium이 분리되었고, 분리된 균주를 대상으로 항균제 내성양상, class I integron 특성 및 pulse-field gel electrophoresis유형(PFGE 유형; pulsotype)이 조사되었다. 35주 모두가 한가지 이상의 항균제에 내성을 나타내었고, 돼지로부터 분리된 대부분의 균주는 ampicillin, chloramphenicol, streptomycin, sulfamethoxazole/trimethoprime, tetracyclin, nalidixic acid에 내성을 나타내었다. 35주를 대상으로 class I, II 및 III integron gene cassette를 검색한 결과, 설사환자로부터 분리된 17주 중 3주가 dhfrX-orfF-aadA2 integron gene cassette을 보유하였고, 돼지로부터 분리된 18주중 11주가 dhfrX-orfF-aadA2 integron gene cassette를, 1주가 aadA2 integron을 보유하였다. 그러나 35주 모두가 class 2 integron gene cassette와 class 3 integron gene cassette는 보유하지 않은 것으로 나타났다. 35주의 PEGE 유형은 5가지로 분류되었으며, 31주가 pulsotype A로 나머지 4주는 pulsotype B, C, D, E형으로 각각 나뉘어졌다. 이러한 결과로 볼 때 경북지역에서 사람과 돼지에서의 S. enterica serovar Typhimurium에 의한 살모넬라중은 몇 종류 유행주의 전파에 의한 것임을 보여줄다. dhfrX-orfF-aadA2 integron gene cassette를 보유한 13주가 pulsotype A, dhfrX-orfF-aadA2 integron gene cassette을 보유한 1주가 pulsotype B, aadA2 integron을 보유한 1주가 pulsotype E, integron을 보유하고 있지 않은 15주가 pulsotype A로 나타났다. Thirty five Salmonella enterica serovar Typhimurium strains were isolated from diarrheic patients and pigs in Gyeongbuk area from 2003 to 2004. All 35 strains (17 strains from diarrheic patients and 18 from pigs) were resistant to more than one drug and most of strains isolated from pigs were resistant to ampicillin, ohloram-phenicol, streptomycin, sulfamethoxazole-trimethoprime, tetracyclin and nalidixic acid. Each isolate was also screened or the presence of class I, II and III integron gene cassettes. Among 35 strains,3 out of 17 strains isolated from diarrheic patients, carried dhfrX-orfF-aadA2 integron gene cassette and among 18 strains isolated from diseased pigs, 11 strains carried dhfrX-orfF-aadA2 integron gene cassette and 1 strain carried aadA2 integron only. But any class II and class II integron gene cassette were not detected in 35 strains. Thirty five strains were divided by five pulsotypes. Thirty one strains out of thirty five were pulsotype A. Among the remaining 4 strains, one each strain belonged to pulsotype B, C, D and pulsotype E. This data of pulsotypes showed that the widespread of pulsotype A, Salmonella enterica serovar Typhimurium in human and pigs in Gyeongbuk area may have been caused by the dissemination of a few epidemic strains in this area. Thirteen strains contain dhfrX-orfF-aadA2 integron gene cassette showed pulsotype A and one strain contains dhfrX-orfF-aadA2 integron gene cassette showed pulsotype B. One strain contains aadA2 integron showed pulsotype E. But fifteen strains do not contain any integron showed pulsotype A.

임상 검체분리 Pseudomonas spp. 및 Acinetobacter spp.에서 검출된 blaVIM-2 카세트 보유 인테그론 구조의 다양성

염종화,신희봉,윤동은,정윤섭 대한임상미생물학회 2012 Annals of clinical microbiology Vol.15 No.4

Background: Metallo-β-lactamase-mediated carbapenem resistance has been increasingly reported in Pseudomonas, Acinetobacter and other Gram-negative bacilli (GNB) in many countries. A few studies showed highly variable structure of MBL-gene cassette-carrying integrons. The aim of this study was to determine the structure of blaVIM-2-carrying integrons in Pseudomonas and Acinetobacter. Methods: blaVIM-2-carrying GNB were isolated at a Korean hospitals during the years 1995-1999 and 2005. The size of blaVIM-2-carrying integrons was estimated by the PCR products. Representative integrons were sequenced by the dideoxy-chain termination method. The MICs of antimicrobial agents were tested by the CLSI agar dilution methods. Results: During the years 1995-1999 and 2005, the approximate size of the blaVIM-2-carrying class 1 integrons was 3-7 kb in 35 Pseudomonas isolates and 3-5 kb in 24 Acinetobacter isolates. The integrons carried one-five resistance gene cassettes in addition to the blaVIM-2 cassette. Other resistance gene cassettes found were blaOXA-1, aacA1, aac(6’)-I, and aac(6’)-II. Interestingly, sequences homologous to part of a putative class II intron were inserted into the recombination site of the last cassette in four of nine integrons. The class 1 integron from P. aeruginosa isolates had fused orf/IntI1 in a downstream leftward inverted repeat (IRi). Conclusion: According to period, the size and structure of blaVIM-2-carrying integrons are quite variable,but an identical one is also present in a different genus,indicating high mobility of the blaVIM-2 cassette and horizontal transfer of the whole integron. We suggest that the class 1 integron containing the blaVIM-2gene is spreading horizontally among Gram- negative bacilli and is undergoing continuous development in Korea.

국내에서 분리된 Acinetobacter baumannii의 Integron과 연관된 다제내성 분석

김성환,최지혜,박은진,서인원,손승렬,Kim, Seong-Hwan,Choi, Ji-Hye,Park, Eun-Jin,Suh, In-Won,Son, Seung-Yeol 한국미생물학회 2010 미생물학회지 Vol.46 No.3

임상분리 균주인 A. baumannii 1625는 imipenem (carbapenem 계열)을 포함한 대부분의 ${\beta}$-lactam계열의 항생제들과 kanamycin, gentamicin, tobramycin 및 제 3세대와 4세대 cephalosporin 계열의 항생제들에 광범위한 내성을 나타내었고, 한국에서는 드문 IMP-1형 metallo-${\beta}$-lactamase (MBL)를 생성하는 균주임이 확인되었다. A. baumannii 1625는 약 2.5 kb 크기의 class 1 integron을 갖고 있었으며, 이 integron내에 aminoglycoside계열 내성 유전자인 accA4, carbapenem 계열 내성 유전자인 $bla_{IMP-1}$, 광범위한 ${\beta}$-lactam 내성 유전자인 $bla_{OXA-2}$ 유전자 cassette들이 차례대로 위치해 있음을 확인하였는데, 이것은 IMP-1형과 OXA-2형 ${\beta}$-lactamase의 유전자를 같은 integron내에 동시에 갖는 새로운 배열 및 구조로서 이전에 국내에서 보고된 바 없는 것이다. 이 2.5 kb 크기의 integron을 항생제 내성이 없는 E. coli에 형질전환 시켰을 때, imipenem, ampicillin, piperacillin, cefazolin, cefoperazone, aztreonam 등의 항생제들에 대하여 8배 이상 증가된 내성정도를 보였다. 이는 A. baumannii 1625의 integron이 다제내성을 부여하는 기능을 하고 있음을 보여준다. Acinetobacter baumannii 1625, a clinical isolate identified by Vitek and 16S rDNA sequence, showed an extended resistance to most ${\beta}$-lactams including imipenem, kanamycin, gentamicin, tobramycin, and cephalosporins of the third and fourth generations, and produced metallo-${\beta}$-lactamase (MBL) of IMP-1 type which is rare in Korea. The isolate contained a class 1 integron of about 2.5 kb in size and the integron included accA4 (aminoglycoside resistance gene), $bla_{IMP-1}$ (carbapenem resistance gene), and $bla_{OXA-2}$ (extended-spectrum ${\beta}$-lactam resistance gene) gene cassettes in order. The coexistence of IMP-1 type and OXA-2 type ${\beta}$-lactamase gene cassettes in an integron has not been reported in Korea. The transformed integron rendered the E. coli transformant resistant more than eight folds against imipenem, ampicilin, piperacillin, cefazolin, cefoperazone, and aztreonam comparing to the reference strain. This study clearly showed that the extended multi-drug resistance of A. baumannii 1625 was mainly due to the integron.

Analysis of a Novel Class 1 Integron Containing Metallo-β-Lactamase Gene VIM-2 in Pseudomonas aeruginosa

Jae Hoon Jeong,신경섭,Jang Won Lee,Eun Jin Park,Seung-Yeol Son 한국미생물학회 2009 The journal of microbiology Vol.47 No.6

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 aadA1 (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.

Distribution and Characterization of Integrons in Enterobacteriaceae Isolates from Chickens in Korea

( Ji Youn Sung ),( Ji Eun Oh ) 한국미생물 · 생명공학회 2014 Journal of microbiology and biotechnology Vol.24 No.7

The use of antimicrobial agents for additives or therapeutics is strongly associated with aprevalence of antimicrobial resistance in commensal Enterobacteriaceae. We aimed to characterize integrons in Enterobacteriaceae isolates obtained from chicken cecums in Korea. Moreover, the correlation between integron gene cassettes and antimicrobial resistance was also investigated. A total of 90 isolates the belonged to Enterobacteriaceae were recovered from chickens grown at Gyeongsang and Chungcheong provinces in Korea. Antimicrobial susceptibility tests were performed by the disk diffusion method. PCR and DNA sequencing were also performed to characterize the gene cassette arrays of the integrons. Of the 90 Enterobacteriaceae isolates tested, 39 (43.3%) and 10 (11.1%) isolates carried class 1 and 2 integrons, respectively. Whereas the class 2 integron did not contain gene cassettes, the class 1 integrons carried seven different gene cassette arrays. The class 1 integrons harbored genes encoding resistant determinants to aminoglycosides (aadA1, aadA2, and aadA5), trimethoprim (dfrA1, dfrA12, dfrA17, and dfrA32), lincosamides (linF), and erythromycin (ereA). Moreover, the presence of a class 1 integron was significantly related to a high resistance rate of antimicrobial agents, such as spectinomycin and trimethoprim. We confirmed that diverse class 1 integrons were widely distributed in Enterobacteriaceae isolates from chickens and directly contributed to the resistance to diverse antimicrobial agents in Korea.

Epidemiological Characterizations of Class 1 Integrons from Multidrug-Resistant Acinetobacter Isolates in Daejeon, Korea

성지연,구선회,김세미,권계철 대한진단검사의학회 2014 Annals of Laboratory Medicine Vol.34 No.4

Background: Multidrug-resistant (MDR) Acinetobacter spp. acquire antimicrobial agent- resistance genes via class 1 integrons. In this study, integrons were characterized to inves- tigate the antimicrobial resistance mechanisms of MDR Acinetobacter isolates. In addi- tion, the relationship between the integron type and integron-harboring bacterial species was analyzed by using epidemiological typing methods. Methods: Fifty-six MDR Acinetobacter spp.- A. baumannii (N=30), A. bereziniae (N=4), A. nosocomialis (N=5), and A. pittii (N=17)-were isolated. The minimum inhibitory con- centrations (MICs) were determined on the basis of the results of the Epsilometer test (Etest). PCR and DNA sequencing was performed to characterize the gene cassette arrays of class 1 integrons. Multilocus sequence typing (MLST) and repetitive extragenic palin- dromic sequence (REP)-PCR were performed for epidemiological typing. Results: Class 1 integrons were detected in 50 (89.3%) of the 56 isolates, but no class 2 or 3 integron was found within the cohorts. The class 1 integrons were classified into 4 types: 2.3-kb type A ( aacA4 - catB8 - aadA1 ), 3.0-kb type B ( aacA4 - blaI MP-1 - bla OXA-2 ), 3.0-kb type C ( bla VIM-2 - aacA7 - aadA1 ), and 1.8-kb type D ( aac3 - 1 - bla OXA-2 - orfD ). Type A was most prevalent and was detected only in A. baumannii isolates, except for one A. bereziniae isolate; however, type B was amplified in all Acinetobacter isolates except for A. bauman- nii isolates, regardless of clone and separation time of the bacteria. Conclusions: Although class 1 integron can be transferred horizontally between unrelated isolates belonging to different species, certain types of class 1 integrons tend to transfer horizontally and vertically among A. baumannii or non- baumannii Acinetobacter isolates.

Spread of multidrug-resistant <i>Escherichia coli</i> harboring integron via swine farm waste water treatment plant

Park, Jin-Hyeong,Kim, Young-Ji,Binn-Kim, Young-Ji,Seo, Kun-Ho Elsevier 2018 Ecotoxicology and environmental safety Vol.149 No.-

<P>Wastewater treatment plants (WWTPs) that release treated wastewater into the environment have emerged as a major threat to public health. In this study, we investigated Escherichia coli load and antibiotic-resistance profiles across different treatment processes at a swine farm WWTP. The frequency of the detection of class 1 and 2 integrons, and their association with antibiotic resistance, were also analyzed. Samples were obtained at each of five sampling sites that represented each processing step within the WWTP. The largest decrease in E. coil load was observed during the anaerobic digestion step (from 4.86 to 2.89 log CFU/mL). Isolates resistant to-lactam antibiotics were efficiently removed after a series of treatment steps, whereas the proportions of isolates resistant to non-lactam antibiotics and multidrug-resistant strains were maintained across treatments. The occurrence of integron-positive strains was not significantly different at the various sampling sites (43.4-70%; p > 0.05). Of the class 1 integron-positive isolates, 17.9% harbored the integron-associated gene cassettes aadA2, aadAl2, aadA22, and dfrA15. To the best of our knowledge, this is the first description of a class 1 integron containing the aadAl2 gene cassette from a swine farm and the presence of a class 1 integron containing dfrA15 in E. coll.. This suggests that novel antibiotic-resistance gene cassette arrays could be generated in swine farm WWTPs. Moreover, 75% of integron-positive strains were categorized as multidrug resistant, whereas only 15.4% of integron-negative strains were multidrug resistant (p < 0.05), indicating that integrons may be responsible for mediating resistance in WWTPs. With regard to the occurrence of multidrug-resistant, integron-positive E. coil recovered from the final effluent, our results highlighted the potential risks associated with wastewater discharge from swine farm WWTPs in terms of the spread of antibiotic-resistant bacteria to the aquatic environment.</P>

Molecular Epidemiology of Integron-Associated Antimicrobial Gene Cassettes in the Clinical Isolates of Acinetobacter baumannii from Northern Taiwan

Ming-Feng Lin,Ming- Li Liou,Chi-Chao Tu,Hui-Wen Yeh,Chung-YuLan 대한진단검사의학회 2013 Annals of Laboratory Medicine Vol.33 No.4

Background: The aims of this study were to understand the molecular epidemiology of integron-associated gene cassettes in Acinetobacter baumannii across four hospitals in northern Taiwan and to clarify the relationship between the presence of integrons and antibiotic-resistant phenotypes. Methods: Sixty-five A. baumannii isolates, collected from the patients of four regional hospitals in northern Taiwan in 2009, were tested for the presence of integrons and their associated gene cassettes. The susceptibility difference between integron-positive and integron-negative A. baumannii strains was analyzed. Antibiotic-resistant phenotypes among A. baumannii with different types of gene cassette array combinations were also compared. Results: Around 72% of the A. baumannii isolates carried class 1 integrase genes. Despite this, only three gene cassette arrays were found in the integrons. Integron-positive strains were significantly more resistant to all the tested antibiotics than the integrase-negative strains. All the four types of A. baumannii with different gene cassette array combinations were multidrug-resistant in nature. Gene cassette array aacA4-catB8-aadA1 existed in all the integron-positive A. baumannii isolates. Repetitive-sequence-based PCR (rep-PCR)results revealed the prevalence of one major cluster of imipenem-resistant A. baumannii strains (84%) in the four regional hospitals. Conclusions: The presence of integrons with associated antimicrobial resistance gene cassettes can be used as a representative marker of multidrug resistance in A. baumannii. Some prevalent gene cassette arrays may exist among epidemiologically unrelated A. baumannii strains.