a Department of Microbiology, College of Medicine, Seonam University, Namwon, Chunpook 590-711; b Department of Microbiology, College of Medicine, University of Dankook, Chonan, Chungpook 330-714; c Department of Microbiology, School of Medicine, Kyungpook National University, Taegu 700-422, Korea
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Abstract |
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Introduction |
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The DHFRs were classified into two families using amino acid sequence analysis. The class A DHFR family includes most of the known trimethoprim-resistant DHFRs. The DHFRs in this family are 6488% identical in amino acids and mediate high-level resistance to trimethoprim.1 dfrA1, dfrA5, dfrA7 and dfrA14 were found to be cassette-associated, although dfrA6 was not found in an identifiable cassette.7 dfrA12 and dfrA13, which are associated with cassettes, are closely related to one another but not to members of this family. The class B DHFR family includes types IIa, IIb and IIc, which are completely unrelated to other DHFRs but which are closely related to one another. Genes that encode the class B DHFRs are all cassette-associated.8 To date, more than 16 DHFRs have been characterized in Gram-negative facultative bacilli.9
Different dfr genes seem to have arisen in different parts of the world and then spread to geographically distant regions. In studies of the epidemiology of trimethoprim-resistant dfr genes in clinical isolates, the most commonly found gene is dfrA1, which is borne on Tn7.5,10 The new dfrA17 gene cassette was recently reported in E. coli and conferred high-level resistance to trimethoprim.11 This gene cassette was 91 and 68% identical to dfrA7 and dfrA1, respectively.
The frequency of resistance to trimethoprim among enterobacterial pathogens has been reported to be higher in Korea than in other countries.12 Little is known about the epidemiology of trimethoprim-resistance-conferring dfr genes in Korea and the dfrA17 gene in the world. In this study, we investigated the prevalence of dfr genes among urinary isolates of E. coli and studied the association of dfr genes with mobile genetic elements in order to determine the transmission mechanisms of dfr genes in Korea.
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Materials and methods |
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A total of 122 E. coli were isolated at Kyungpook National University Hospital, Taegu, Korea during the period 1994 1996. The isolates were obtained from urine specimens from patients with significant bacteriuria (>105 cfu/mL). Seventy-seven of the 122 isolates were selected according to their resistance to trimethoprim (MIC 16 mg/L).
Antibiotic susceptibility testing
The MICs of the antibiotics were determined by agar dilution in MuellerHinton agar (MHA) (Difco Laboratories, Detroit, MI, USA) with a Steers multiple inoculator13 according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS).14 The MIC was defined as the lowest concentration of antibiotic that completely inhibited the growth of the organism. E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains. The antibiotics included were trimethoprim (Sigma, St Louis, MO, USA), ampicillin, amikacin, gentamicin, kanamycin, streptomycin, tobramycin, sulphamethoxazole and ciprofloxacin. For antibiogram analysis of transconjugants, the same method as that used for the MIC determinations was used, except that a fixed concentration of antibiotics was incorporated into MHA.
Transfer of resistance determinants and plasmid analysis
E. coli RG488 Rifr and RG176 Nalr were used as recipients for conjugation experiments. Logarithmic phase of donor and the recipient strains were grown in trypticase soy broth (Difco Laboratories), and were mixed and incubated at 37°C for 20 h. Transconjugants were selected on MHA medium supplemented with trimethoprim (16 mg/L) and nalidixic acid (50 mg/L) or rifampicin (50 mg/L). To confirm the presence of plasmids and to estimate their sizes, plasmids from clinical isolates and their transconjugants were isolated by the method of Birnboim & Doly,15 and analysed by electrophoresis in 0.7% agarose gels.
PCR amplification of the dfr genes
The PCR was performed in a total volume of 20 µL containing the following: 2 µL of boiled bacterial suspensions, 50 pM of each primer, 250 µM of dNTP, 10 mM TrisHCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 and 1.5 U of Taq DNA polymerase (TaKaRa Shuzo Co. Ltd, Shiga, Japan). The primers D1 and D2 described previously by Gibrel & Sköld16 were used for the detection of dfrA1. Four primer pairs were designed according to the published sequences of dfr genes (Table I). To detect each dfr gene, the PCR products of dfrA14 with the primers D3 and D4, dfrA13 with the primers D7 and D8, and dfrA17 with the primers D9 and D10 possessed a restriction site for EcoRI, EcoRV and PstI (Promega, Madison, WI, USA), respectively. The amplification reaction with a DNA thermal cycler (Perkin-Elmer Cetus) consisted of 30 cycles of denaturation at 94°C for 30 s, various annealing temperatures (58°C for primers D1 and D2, 54°C for primers D3 and D4, 57°C for primers D5 and D6, 58°C for primers D7 and D8, and 51°C for primers D9 and D10) for 30 s, and extension at 72°C for 1 min. The PCR was ended with a final elongation step of 5 min at 72°C.
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To amplify class 1 integrons and the intI2 gene of Tn7, primer pairs 5'-CS (5'-GGCATCCAAGCAGCAAG-3') and 3'-CS (5'-AAGCAGACTTGACCTGA-3'), and Int2A and Int2B were used, respectively.17 The primers Int2A (5'-ATGTCTAACAGTCCATTTTTAAATTCTA-3') and Int2B (5'-AAATCTTTAACCCGCAAACGC-3') are located within the intI2 gene of Tn7. The PCR was performed as described previously.17
Southern hybridization
After agarose gel electrophoresis of PCR products of the class 1 integrons, denatured DNAs were transferred on to a positively charged nylon membrane (Hybond-N+; Amersham, Braunschweig, Germany) by the capillary method.18 For hybridization assays the DIG DNA labelling and detection kit (Boehringer Mannheim, Mannheim, Germany) was used according to the manufacturer's instructions. The hybridization procedures were performed under high-stringency conditions.
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Results |
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Of the 122 isolates tested, 77 (63.1%) isolates were resistant to trimethoprim. The MICs of the 77 trimethoprim-resistant isolates were found to be 1024 mg/L. In these strains, 76 (98.7%), 72 (93.5%) and 21 (27.3%) isolates were resistant to ampicillin, sulphamethoxazole and ciprofloxacin, respectively. Sixty-five (84.4%) isolates were resistant to more than one aminoglycoside.
Identification of the trimethoprim-resistant dfr genes
Among the 77 trimethoprim-resistant isolates, dfrA1, dfrA5, dfrA7, dfrA12 and dfrA17 were detected (Figure 1a). dfrA7 and dfrA17 were co-amplified by PCR with the primers D9 and D10. The PCR products of dfrA17 possessed a restriction site for PstI and digested into 134 and 61 bp fragments (Figure 1b
). Overall, 75 dfr genes were detected in 72 isolates: dfrA17 was detected in 27 isolates, dfrA12 in 26 isolates, dfrA1 in 15 isolates, dfrA5 in four isolates and dfrA7 in three isolates (Table II
). Two different dfr genes co-existed in three isolates (dfrA1 and dfrA17, dfrA7 and dfrA12, and dfrA1 and dfrA12). None of the isolates was found to have dfrA8, dfrA13 or dfrA14. Five isolates in this study showed a negative result when tested for the presence of dfr genes.
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To investigate the association of dfr genes with integrons, we amplified the class 1 integrons and determined whether dfr genes were inserted between the conserved segments of the class 1 integrons. The class 1 integrons of the E. coli isolates that carried dfrA1, dfrA5, dfrA7, dfrA12 and dfrA17 were amplified in lengths of approximately 1900, 720, 770, 1900 and 1660 bp, respectively (Figure 2). All isolates that carried the known dfr genes were also found to possess the class 1 integrons, with the exception of seven (four isolates carrying dfrA17, two isolates carrying dfrA1 and one isolate carrying dfrA5). The seven isolates that carried the known dfr genes but were negative in PCR amplification of the class 1 integrons were studied in detail by a PCR mapping analysis.19 The PCR products of 275 bp were amplified by using the primers 5'-CS and D10 (downstream primer of dfrA17) in four isolates. The PCR amplification with the upstream primer of dfrA1 and dfrA5 and 3'-CS was positive in 1/2 and 1/1 isolates, respectively. In Southern blot hybridization of PCR products of the class 1 integrons with each dfr gene probe, all of the amplified products of E. coli isolates that carried dfrA5, dfrA7, dfrA12 and dfrA17 were hybridized to each specific dfr gene probe (Table II
). Using Southern blot and PCR mapping analysis, dfrA1 inserted as a gene cassette in class 1 integrons was found in 10 of the 15 isolates. The intI2 gene of Tn7 was amplified in two out of five isolates.
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In order to test the transferability of trimethoprim resistance in the isolates, a conjugation experiment was carried out with all of the isolates with the known dfr genes. Trimethoprim resistance was conjugally transferable to a recipient E. coli strain in 31 (43.1%) of 72 isolates. Of the transconjugant strains that transferred trimethoprim resistance, dfrA12, dfrA17 and dfrA1 were transferred at a frequency of 14/26, 12/27 and 6/15, respectively (Table II). Plasmid profiles of each wild E. coli strain and its transconjugant showed that the transconjugants acquired plasmids in the range 67157 kb (Table III
).
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Discussion |
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We investigated the prevalence of dfr genes in E. coli isolated from urinary tract infections. Our data demonstrated that dfrA17 and dfrA12 were the most prevalent genes, and that dfrA1, dfrA5 and dfrA7 were also detected. This prevalence of dfr genes differed from all other previous data. In a report of the prevalence of trimethoprim resistance genes in Gram-negative commensal faecal flora from South Africa, the most prevalent gene was dfrA14, followed by dfrA7, dfrA1, dfrA8, dfrA13, dfrA5 and dfrA12.23 In a report from the UK, dfrA1 was the most frequent gene in urinary tract isolates.24 dfrA1 was initially identified as a component of Tn7 and it was thought that the widespread dissemination of the gene was caused by Tn7.9 However, there is evidence that dfrA1 forms a gene cassette that is not associated with Tn7. Recently, dfrA1 that is integrated in gene cassettes seems to be more widespread than those that bear Tn7.24 In this study, dfrA1 was inserted as a gene cassette in 10 of the 15 isolates, and Tn7 was detected in two out of five isolates. All of the dfrA5, dfrA7, dfrA12 and dfrA17 genes were found to be associated with gene cassettes. This result suggests that dfr genes inserted in integrons have spread efficiently under the selective pressure of the widespread use of trimethoprim.
When dfr genes become part of a gene cassette, they acquire the ability to move readily from one genome to another.8 The plasmid-borne dfr gene cassettes in Gram-negative bacteria can move to other bacteria by conjugation. We demonstrated that dfrA1, dfrA12 and dfrA17 were experimentally transferred to a recipient E. coli in 31 of the 72 trimethoprim-resistant isolates. This suggests that an increasing proportion of dfr genes inserted in integrons provides an important mechanism for the dissemination of these genes.
In summary, dfrA17 and dfrA12 inserted in integrons are the most prevalent genes in the urinary isolates of E. coli from Korea, and dfrA1, dfrA12 and dfrA17 in integrons were transferred to recipient bacteria through conjugative R plasmids in 43.1% of E. coli isolates. This result suggests that the dfr genes inserted in integrons may have important implications for the further dissemination of trimethoprim resistance.
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Acknowledgments |
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Notes |
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References |
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2 . Burchall, J. J. & Hitchings, G. H. (1965). Inhibitor binding analysis of dihydrofolate reductases from various species. Molecular Pharmacology 1, 12636.[Abstract]
3 . Amyes, S. G. B. & Smith, J. T. (1974). R-factor trimethoprim resistance mechanism: an insusceptible target site. Biochemical and Biophysical Research Communications 58, 4128.[ISI][Medline]
4 . Jansson, C., Franklin, A. & Sköld, O. (1992). Spread of a newly found trimethoprim resistance gene, dhfrIX, among porcine isolates and human pathogens. Antimicrobial Agents and Chemotherapy 36, 27048.[Abstract]
5 . Steen, R. & Sköld, O. (1985). Plasmid-borne or chromosomally mediated resistance by Tn7 is the most common response to ubiquitous use of trimethoprim. Antimicrobial Agents and Chemotherapy 27, 9337.[ISI][Medline]
6
.
Adrian, P. V., Du Plessis, M., Klugman, K. P. & Amyes, S. G. B. (1998). New trimethoprim-resistant dihydrofolate reductase cassette, dfrXV, inserted in a class 1 integron. Antimicrobial Agents and Chemotherapy 42, 22214.
7 . Hall, R. M. & Collis, C. M. (1998). Antibiotic resistance in gram-negative bacteria: the role of gene cassettes and integrons. Drug Resistance Updates 1, 10919.[ISI]
8 . Recchia, G. D. & Hall, R. M. (1995). Gene cassettes: a new class of mobile element. Microbiology 141, 301527.[ISI][Medline]
9 . Then, R. L. (1993). History and future of antimicrobial diaminopyrimidines. Journal of Chemotherapy 5, 3618.[ISI][Medline]
10 . Heikkilä, E., Sundström L., Skurnik, M. & Huovinen, P. (1991). Analysis of genetic localization of the type I trimethoprim resistance gene from Escherichia coli isolated in Finland. Antimicrobial Agents and Chemotherapy 35, 15629.[ISI][Medline]
11 . White, P. A., Mclver, C. J., Deng, Y. & Rawlinson, W. D. (2000). Characterisation of two new gene cassettes, aadA5 and dfrA17. FEMS Microbiology Letters 182, 2659.[ISI][Medline]
12 . Seol, S. Y., Chang, H. K., Kim, J. M., Shin, H. S., Lee, J. C., Lee, Y. C. et al. (1997). Molecular epidemiologic analysis of nosocomial Escherichia coli isolates. Journal of the Korean Society for Microbiology 32, 114.
13 . Steers, E., Flotz, E. L., Gravis, B. S. & Riden, J. (1959). Inocular replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiotica et Chemotherapica 9, 30711.
14 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFourth Edition: Approved Standard M7-A4. NCCLS, Villanova, PA.
15 . Birnboim, C. & Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7, 151323.[Abstract]
16
.
Gibrel, A. & Sköld, O. (1998). High-level resistance to trimethoprim in clinical isolates of Campylobacter jejuni by acquisition of foreign genes (dfr1 and dfr9) expressing drug-insensitive dihydrofolate reductases. Antimicrobial Agents and Chemotherapy 42, 305964.
17
.
Tosini, F., Visca, P., Luzzi, I., Dionisi, A. M., Pezzella, C., Petrucca, A. et al. (1998). Class 1 integron-borne multiple-antibiotic resistance carried by IncFI and IncL/M plasmids in Salmonella enterica serotype typhimurium. Antimicrobial Agents and Chemotherapy 42, 30538.
18 . Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Analysis and cloning of eukaryotic genomic DNA. In Molecular Cloning: A Laboratory Manual, 2nd edn, (Sambrook, J., Fritsch, E. F. & Maniatis, T., Eds), pp. 9.349.51. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
19 . Levesque, R., Piche, L., Larose, C. & Roy, P. H. (1995). PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrobial Agents and Chemotherapy 39, 18591.[Abstract]
20 . Lamikanra, A. & Ndep, R. B. (1989). Trimethoprim resistance in urinary tract pathogens in two Nigerian hospitals. Journal of Antimicrobial Chemotherapy 23, 1514.[Abstract]
21 . Murray, B. E., Alvarado, T., Kim, K. H., Vorachit, M., Jayanetra, P., Levine, M. M. et al. (1985). Increasing resistance to trimethoprim-sulfamethoxazole among isolates of Escherichia coli in developing countries. Journal of Infectious Diseases 152, 110713.[ISI][Medline]
22 . Urbina, R., Prado, V. & Canelo, E. (1989). Trimethoprim resistance in enterobacteria in Chile. Journal of Antimicrobial Chemotherapy 23, 1439.[Abstract]
23 . Adrian, P. V., Klugman, K. P. & Amyes, S. G. B. (1995). Prevalence of trimethoprim resistant dihydrofolate reductase genes identified with oligonucleotide probes in plasmids from South Africa isolates of commensal faecal flora. Journal of Antimicrobial Chemotherapy 35, 497508.[Abstract]
24 . Towner, K. J., Brennan, A., Zhang, Y., Holtham, C. A., Brough, J. L. & Carter, G. I. (1994). Genetic structures associated with spread of the type Ia trimethoprim-resistant dihydrofolate reductase gene amongst Escherichia coli strains isolated in the Nottingham area of the United Kingdom. Journal of Antimicrobial Chemotherapy 33, 2532.[Abstract]
Received 13 July 2000; returned 20 November 2000; revised 2 January 2001; accepted 30 January 2001