Prevalence of dfr genes associated with integrons and dissemination of dfrA17 among urinary isolates of Escherichia coli in Korea

Hak Sun Yu1, Je Chul Lee2, Hee Young Kang1, Young Sook Jeong1, Eun Young Lee1, Chul Hee Choi1, Seong Ho Tae1, Yoo Chul Lee1, Sung Yong Seol1 and Dong Taek Cho1,*

1 Department of Microbiology, School of Medicine, Kyungpook National University, 101, Dongin-dong, Jung-gu, Daegu, 700-422; 2 Department of Microbiology, Eulji University School of Medicine, Taejeon, 301-832, Korea

Received 23 May 2003; returned 29 October 2003; revised 19 November 2003; accepted 4 December 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The association of trimethoprim-resistant dfr genes with integrons was investigated in urinary Escherichia coli isolates in Korea from the last two decades.

Methods: Of 623 E. coli isolates from urine specimens, 421 trimethoprim-resistant isolates were studied for dfr genes associated with integrons. Integrase genes were amplified and the PCR products restricted using HinfI to classify integron types. Gene cassette regions for the class 1 and class 2 integrons were amplified and sequenced. PFGE was performed to determine the epidemiological relationship of E. coli isolates.

Results: The carriage of class 1 integrons was found to be significantly higher in trimethoprim-resistant isolates (69%) than in trimethoprim-susceptible isolates (19%). Among the trimethoprim-resistant isolates, the frequency of dfr genes associated with class 1 integrons increased sharply from 10% of the isolates during 1980–1985 to 53% during 1996–1997 and to 46% during 2001–2002. Five different dfr cassettes—dfrA1, dfrA5, dfrA7, dfrA12 and dfrA17—were identified among the urinary E. coli isolates from the last two decades; dfrA12 was the most prevalent during 1980–1985 and dfrA17 during 1996–1997 and 2001–2002. The majority of dfr genes associated with class 1 integrons were conjugally transferable to recipient E. coli strains. The E. coli isolates that carried dfrA17 associated with class 1 integrons were found to be phylogenetically unrelated, indicating that dfrA17 was widely distributed in the different clones of E. coli.

Conclusion: Class 1 integrons were found to be an important genetic element of resistance to trimethoprim among urinary E. coli in Korea, and the prevalence of dfrA17 was mainly due to the horizontal transfer of class 1 integrons through conjugative plasmids.

Keywords: trimethoprim, E. coli, resistance, conjugative plasmids


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Integrons are genetic elements that have the ability to integrate or mobilize gene cassettes by site-specific recombination.15 Gene cassettes, mostly encoding antibiotic resistance, consist of a gene flanked by an integrase-specific recombination site, known as a 59-base element.69 Since many antibiotic resistance genes are part of a gene cassette in Gram-negative bacteria, integrons have been found to play an important role in the carriage and dissemination of antibiotic resistance genes.10 Based on the sequences of integrase genes, four classes of integrons have been described.10,11 Class 1, 2 and 3 integrons are known to be associated with antibiotic resistance, whereas class 4 integrons are a distinctive class found in Vibrio cholerae and are not associated with antibiotic resistance.12 Class 1 integrons are most commonly found in clinical isolates of Gram-negative bacteria and are strongly associated with multiple antibiotic resistance.13,14 To date, more than 60 distinct gene cassettes have been identified.8 Class 2 integrons include Tn7 and its relatives, and are associated with resistance to trimethoprim, streptomycin and spectinomycin.8

Escherichia coli isolates from clinical specimens are mostly resistant to multiple antibiotics and a substantial proportion of E. coli isolated from the urinary tract is resistant to trimethoprim.1517 The most commonly encountered resistance mechanism is the acquisition of a trimethoprim-resistant foreign dfr gene through mobile genetic elements, including plasmids or transposons,1821 which has led to the rapid emergence of trimethoprim resistance among bacterial populations. At present, 17 different types of trimethoprim-resistant dfr genes have been identified in Gram-negative bacteria, and nine dfr gene cassettes, including dfrA1, dfrA5, dfrA7, dfrA12, dfrA14, dfrA17, dfrB1, dfrB2 and dfrB3, have been found in class 1 integrons.10,22

The most widespread dfr gene among Gram-negative bacteria isolated in the 1970s and 1980s was dfrA1, which was attributed to the rapid spread of Tn7.20,23 However, in our previous study, dfrA12 and dfrA17 were the most prevalent in 1994 in urinary E. coli isolates from Korea.15 Both these dfr genes are associated with class 1 integrons, and the dfr genes inserted in integrons seem to be more widespread than those that are not so inserted.17 However, little is yet known about changes in dfr genes associated with class 1 integrons over the course of time. This study investigated the prevalence of different dfr genes associated with integrons among urinary E. coli isolates from one Korean hospital during the last two decades. Furthermore, the dissemination mechanism of dfrA17, the most prevalent dfr gene in recent years, was characterized.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

A total of 623 E. coli, including 243 isolates from the period 1980–1985, 179 from 1996–1997 and 201 from 2001–2002, were isolated at Kyungpook National University Hospital, Daegu, Korea. The isolates were obtained from urine specimens from patients with significant bacteriuria (>105 cfu/mL).

Determination of trimethoprim resistance

The MICs of trimethoprim were determined by agar dilution in a Mueller–Hinton agar (MHA) medium (Difco Laboratories, Detroit, MI, USA) using a Steers multiple inoculator24 according to the guidelines of the NCCLS.25 The inoculated plates were incubated at 37°C for 20 h, and the MIC defined as the lowest concentration of trimethoprim that completely inhibited the growth of the organism. E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains. Trimethoprim-resistant isolates were selected according to their resistance to trimethoprim (MIC >= 16 mg/L), and 421 trimethoprim-resistant isolates were studied further.

Transfer of trimethoprim-resistance determinants

E. coli RG488 Rifr and RG176 Nalr were used as the recipients for the conjugation experiment. The donor and recipient strains in a logarithmic phase were grown in a trypticase soy broth (TSB; Difco Laboratories), mixed and incubated at 37°C for 20 h. The transconjugants were then selected on an MHA medium supplemented with trimethoprim (50 mg/L) and nalidixic acid (50 mg/L) or rifampicin (50 mg/L).

Genomic and plasmid DNA isolation and Southern hybridization

E. coli was inoculated into 4 mL of TSB and incubated at 37°C for 20 h with shaking. The bacteria were centrifuged at 12 000 rpm for 5 min, the supernatant removed and the pellet suspended in a TE buffer (25 mM Tris, pH 8.0, 10 mM EDTA) supplemented with 50 mM dextrose. The plasmid DNA was isolated using the alkaline extraction method,26 and the genomic DNA isolated as described previously.27 The extracted DNA was separated by electrophoresis on 0.7% agarose gels. After agarose gel electrophoresis of the DNA, the denatured DNA was transferred onto a positively charged nylon membrane (Hybond-N+; Amersham, Braunschweig, Germany) using the capillary method.28 For the hybridization assays, a DIG DNA labelling and detection kit (Boehringer Mannheim, Mannheim, Germany) was used according to the manufacturer’s instructions. The purified integrase gene from the PCR products was used as the probe DNA.

PCR amplification of integrase genes

The PCR was performed in a total volume of 50 µL containing the following: 2 µL of boiled bacterial suspensions, 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.2 µM of each primer and 1 U of Taq polymerase. The template was prepared by suspending a loopful of each isolate, which had been growing on a trypticase soy agar (Difco Laboratories) plate, in 200 µL of sterile water, followed by boiling for 10 min and centrifuging for 5 min. The primers specific for the integrase gene are described elsewhere.29 The PCR reaction consisted of an initial denaturation step at 95°C for 1 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and an extension at 72°C for 45 s. To determine the types of integron, the PCR products were restricted using HinfI and analysed according to their restriction fragment length polymorphism.29

Amplification and sequencing of gene cassette regions

The gene cassette regions for the class 1 and class 2 integrons were amplified using primer pairs hep58-hep59 and hep74-hep51, respectively, as described previously.29 To determine identical arrays of cassette genes, the same sized amplicons were restricted with RsaI and HinfI. The PCR products of the gene cassette regions were ligated with a pGEM T-easy vector (Promega, Madison, WI, USA) and introduced into E. coli DH5{alpha} cells. Sequencing reactions were then performed with a double-stranded plasmid preparation using dideoxy chain termination with T7 and Sp6 primers.

PFGE

The genomic DNA was digested with XbaI (Boehringer Mannheim, Mannheim, Germany) for 18 h and separated on a 1.0% agarose gel using a contour-clamped homogeneous-field apparatus (CHEF DRIII systems, Bio-Rad Laboratories, Hercules, CA, USA) in a 0.5 X TBE buffer. The conditions for electrophoresis were 6 V/cm for 20 h with an increasing pulse time of 5–40 s. A {lambda}DNA ladder consisting of 48.5 kb concatemers (Bio-Rad Laboratories) was used as the size standard. The PFGE patterns were interpreted using the criteria established by Tenover et al.30


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Trimethoprim resistance of E. coli isolates

Of the 623 E. coli isolates tested, 421 isolates were resistant to trimethoprim (Table 1). The frequency of trimethoprim resistance was 74% (181/243) in the isolates from the period 1980–1985, 64% (114/179) from 1996–1997 and 63% (126/201) from 2001–2002. As such, the overall frequency of trimethoprim resistance has decreased gradually in the E. coli isolates over the last two decades.


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Table 1. Incidence of integrase genes in urinary E. coli isolates from Korea
 
Detection of integrase genes

To determine whether E. coli isolates carried integrons, the conserved regions of the integrase genes were amplified and the PCR products further restricted using HinfI to determine the types of integron (Figure 1). intI1 was detected in 63% (114/181), 78% (89/114) and 69% (87/126) of the trimethoprim-resistant isolates from 1980–1985, 1996–1997 and 2001–2002, respectively (Table 1), whereas intI2 was detected in 10, five and four of the trimethoprim-resistant isolates from 1980–1985, 1996–1997 and 2001–2002, respectively. Both class 1 and class 2 integrons were detected in eight isolates, comprising five from the 1980s, one from the 1990s and two from the 2000s.



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Figure 1. PCR amplification of integrase genes and restriction fragment length polymorphism analysis by HinfI. A 491 bp PCR product for intI2 possessed a restriction site for HinfI, which was digested into 334 bp and 157 bp. Lane M, 100 bp DNA ladder; lanes 1 and 2, intI1, lanes 3 and 4, intI2; lanes 5 and 6, intI1 and intI2.

 
No class 3 integrons were detected. In total, 290 (69%) intI1 and 19 (5%) intI2 were found in the 421 trimethoprim-resistant isolates. Among the 202 trimethoprim-susceptible isolates, intI1 was detected in 19% (12/62), 17% (11/65) and 20% (15/75) of the isolates from 1980–1985, 1996–1997 and 2001–2002, respectively. As such, class 1 integron carriage was higher in the trimethoprim-resistant isolates (69%) than in the trimethoprim-susceptible isolates (19%).

Association of dfr genes with class 1 integrons

To investigate the association of dfr genes with class 1 integrons, the gene cassette regions were amplified and sequenced. Among the 290 trimethoprim-resistant E. coli isolates carrying intI1, 137 (47%) isolates carried dfr genes in class 1 integrons. dfr genes associated with integrons were found in only 15% (28/181) of the trimethoprim-resistant isolates from 1980–1985, but in 56% (64/114) from 1996–1997 and 48% (61/126) from 2001–2002 (Table 2). As such, the frequency of dfr genes associated with class 1 integrons has increased sharply in the E. coli isolates over the last two decades.


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Table 2. Frequency of dfr genes associated with integrons in trimethoprim-resistant E. coli isolates from Korea
 
Characterization of dfr genes associated with integrons

Five dfr genes—dfrA1, dfrA5, dfrA7, dfrA12 and dfrA17—were detected in seven different class 1 integrons (Figure 2). Three class 1 integrons that carried dfrA1 were amplified to lengths of ~1200, 1500 and 3000 bp (Figure 2), whereas the gene cassette regions that carried dfrA5, dfrA7, dfrA12 and dfrA17 were amplified to lengths of 720, 770, 1900 and 1660 bp, respectively. Only two different dfr genes—dfrA12 and dfrA1—were found in the isolates from 1980–1985, whereas five different dfr genes—dfrA1, dfrA5, dfrA7, dfrA12 and dfrA17—were found in the isolates from 1996–1997 (Table 3). dfrA12 was most prevalent in the isolates from 1980–1985, whereas dfrA17 was most prevalent from 1996–1997 and 2001–2002. Four dfr genes were found in 58 isolates from 2001–2002: dfrA17 was found in 29 isolates, dfrA12 in 22, dfrA5 in five and dfrA1 in four. Two isolates from 2001–2002 carried two different class 1 integrons (Figure 2). All class 2 integrons carried the same gene cassettes as those found in Tn7, namely dfrA1, sat1 and aadA1.



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Figure 2. PCR amplification of gene cassette regions in E. coli isolates. Lanes 1–3, PCR products of class 1 integrons carrying dfrA1; lane 4, PCR product of class 1 integrons carrying dfrA5; lane 5, PCR product of class 1 integrons carrying dfrA7; lane 6, PCR product of class 1 integrons carrying dfrA12; lane 7, PCR product of class 1 integrons carrying dfrA17; lane 8, PCR products of class 1 integrons carrying dfrA5 and dfrA12; lane 9, PCR products of class 1 integrons carrying dfrA12 and dfrA17; and lane 10, PCR product of class 2 integrons carrying dfrA1, sat1 and aadA1.

 

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Table 3. Prevalence of dfr genes associated with class 1 integrons in trimethoprim-resistant E. coli isolates from Korea
 
Transferability of dfr genes associated with integrons

To determine the transferability of the dfr genes associated with class 1 integrons, a conjugation experiment was carried out. Of the 137 dfr genes associated with class 1 integrons tested, 97 (71%) were conjugally transferable. The transfer rates of dfrA1, dfrA5, dfrA7, dfrA12 and dfrA17 were 85% (11/13), 86% (6/7), 100% (1/1), 73% (45/62) and 64% (34/53), respectively (Table 3). The transfer rate of dfrA17 was the lowest among the five dfr genes tested. To determine the genetic localization of the non-transferable dfr genes associated with class 1 integrons, Southern hybridization with an intI1 probe was performed, which revealed that all the dfr genes associated with class 1 integrons were located in plasmids, except for two class 1 integrons carrying dfrA1 and dfrA12 that were located in the chromosome.

Epidemiological typing of E. coli isolates carrying dfrA17

To determine whether the high prevalence of dfrA17 during 1996–1997 and 2001–2002 was caused by the spread of a specific clone in the hospital environment or the horizontal transfer of class 1 integrons between Gram-negative bacteria, 24 isolates that carried dfrA17 were selected and their epidemiological relationship analysed according to their PFGE profiles. Twelve isolates were selected from 1996–1997 and 2001–2002, respectively. Twenty-two of the E. coli isolates exhibited different PFGE patterns with five or more band differences, indicating that they were phylogenetically unrelated based on the criteria established by Tenover et al.30 (Figure 3), whereas two of the E. coli isolates (96K057 and 96K122) were 87% linked based on their PFGE profiles. Accordingly, the widespread association of dfrA17 with class 1 integrons in the different clones of E. coli suggests that the high prevalence of dfrA17 was mainly due to the horizontal transfer of class 1 integrons through conjugative plasmids.



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Figure 3. Dendrogram showing cluster analysis results for 24 E. coli strains carrying dfrA17 associated with class 1 integrons using PFGE profiles with XbaI.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Trimethoprim is a broad-spectrum antimicrobial agent active against enteric pathogens, such as E. coli and Shigella species. Although the resistance rate to trimethoprim among urinary E. coli isolates has slightly decreased during the last two decades, it is still higher in Korea than in some industrialized countries.17,31,32 Furthermore, since trimethoprim alone or in combination with sulfamethoxazole has been commonly used to treat shigellosis in Korea during the last two decades, more than 96% of the Shigella sonnei isolates from Korea during the last two decades are resistant to trimethoprim.33 The reason for the discrepancy in the trimethoprim resistance rate between E. coli and S. sonnei is possibly because the use of trimethoprim in treating urinary tract infections decreased in Korea during the 1990s, due to the high frequency of trimethoprim resistance among urinary isolates of Enterobacteriaceae, as reported in Korea and elsewhere.1517

Among the 623 E. coli isolates tested, 328 (53%) carried class 1 integron structures, as detected by a PCR specific for the integrase gene. White et al.22 reported a similar result in urinary E. coli isolates from Australia, where 44 (49%) isolates carried class 1 integrons among 90 E. coli isolates from 1998–1999. Other studies have also shown the prevalence of class 1 integrons in E. coli isolates from various clinical specimens, for example, 52% in Taiwan and 60% in European countries.34 In this study, class 1 integrons were more prevalent in the trimethoprim-resistant isolates (69%) than in the trimethoprim-susceptible isolates (19%). Although there has been no previous study on the prevalence of class 1 integrons in trimethoprim-resistant bacteria, it is known that class 1 integron-carrying enterobacteria are more likely to be resistant to trimethoprim, sulfamethoxazole, aminoglycosides, quinolones, chloramphenicol, tetracycline and some ß-lactam compounds than integron-negative enterobacteria.34,35 Consequently, when taking all these data together, it would seem that class 1 integrons are widely disseminated in multiresistant Enterobacteriaceae and directly contribute to the resistance to certain antimicrobial agents.

Among the trimethoprim-resistant E. coli isolates, the frequency of dfr genes associated with class 1 integrons increased sharply from 10% in the isolates from 1980–1985 to 53% from 1996–1997 and 46% from 2001–2002, although the rate of class 1 integron carriage did not significantly increase. This suggests that class 1 integrons are an important genetic element for resistance to trimethoprim among urinary isolates of E. coli in Korea. The increase in dfr genes associated with class 1 integrons also raised the question of whether this was due to the emergence of new class 1 integrons carrying dfr genes, or the wide dissemination of class 1 integrons carrying dfr genes through plasmids or transposons. Five different dfr gene cassettes—dfrA1, dfrA5, dfrA7, dfrA12 and dfrA17—were detected in the E. coli isolates. dfrA12 was found to be most prevalent in the isolates from 1980–1985, meanwhile, although dfrA17 was not detected in the isolates from 1980–1985, it was most prevalent from 1996–1997 and 2001–2002. In a previous study by the current authors, dfrA17 and dfrA12 were identified as the two prevalent dfr genes in urinary E. coli isolates from the same hospital during 1994–1996.15 Accordingly, it would seem that urinary E. coli isolates have selected gene cassettes carrying dfr genes, thereby accounting for the high prevalence of dfrA17 in recent years. Moreover, the prevalence of aminoglycoside-resistant genes associated with class 1 integrons has also changed over the course of time, where aadA, aadA2 and aadA5 were found to be the most prevalent genes in isolates from 1980–1985, 1996–1997 and 2001–2002, respectively (data not shown). Seven different class 1 integrons carrying dfr genes were detected in the E. coli isolates tested (Figure 2). Class 1 integrons carrying a single gene cassette, such as dfrA1, dfrA5 or dfrA7, were detected in nine E. coli isolates, whereas the majority of the isolates carried two or more gene cassettes, such as dfrA12-aadA2, dfrA17-aadA5, aacA6-catB4-dfrA1-unknown ORF and dfrA1-aadA2. Overall, a class 1 integron composed of dfrA12-aadA2 was found to be most prevalent in the E. coli isolates from the last two decades.

A conjugation experiment and Southern hybridization were carried out to determine the genetic localization of class 1 integrons. The results revealed that all the isolates, except for two, carried their class 1 integrons in plasmids. Moreover, 71% of the dfr genes associated with class 1 integrons were transferred to recipient strains (Table 3), indicating that most class 1 integrons are horizontally transferred through conjugative plasmids. In a previous study by the current authors, two prevalent dfr genes—dfrA17 and dfrA12—were associated with class 1 integrons, and only half of the genes were transferred to a recipient E. coli,15 whereas in this study, 64% of the class 1 integrons carrying dfrA17 were transferred to recipient strains. Furthermore, the E. coli isolates that carried dfrA17 in class 1 integrons were found to be genetically unrelated, which was also verified by their PFGE profiles (Figure 3), indicating that the horizontal transfer of dfrA17 associated with class 1 integrons through conjugative plasmids was responsible for the wide dissemination of dfrA17 and high prevalence of dfrA17 in the urinary isolates of E. coli.

In conclusion, this study demonstrated that class 1 integrons are an important genetic element for resistance to trimethoprim among urinary E. coli isolates, and that the selection of a specific dfr gene occurs in E. coli isolates over the course of time. The wide dissemination of dfrA17 in urinary E. coli isolates is mainly due to the horizontal transfer of class 1 integrons through conjugative plasmids. Accordingly, integron surveillance could be used to monitor the spread of specific antimicrobial resistance genes in a hospital environment.


    Acknowledgements
 
This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (02-PJ1-PG3-20201-0005).


    Footnotes
 
* Corresponding author. Tel: +82-53-420-6951; Fax: +82-53-427-5664; E-mail: dtcho{at}knu.ac.kr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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