Department of Microbiology, School of Medicine, Kaohsiung Medical University, 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan, Republic of China
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Abstract |
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Introduction |
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Materials and methods |
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E. coli EC107 was isolated from a clinical urine specimen from Kaohsiung Medical University Hospital in Taiwan.
Antimicrobial susceptibility
The susceptibility of E. coli EC107 to antimicrobial agents was determined by the disc agar diffusion method according to the recommendations of the NCCLS.2 The MICs of antimicrobial agents were determined by an agar dilution technique using MuellerHinton agar (Difco, Detroit, MI, USA).3
Plasmid isolation, hybridization and conjugation
Plasmid DNA was extracted from E. coli EC107 by the method described by Kado & Liu.4 Hybridization was performed with an oligonucleotide probe (5'-GCCTCGACTTCGCTGCTGCCC-3') specific for the intI1 gene.5 The conjugation experiment was performed by mixing the donor strain EC107 and the recipient strain, E. coli K-12 20R764 (F lac+ rif r), followed by overnight incubation at 37°C and 28°C.
PCR amplification and nucleotide sequencing
The variable region(s) of integron(s) in EC107 were amplified by using PCR with primers specific for the integron 5' conserved segment (5'-CS; 5'-GGCATCCAAGCAGCAAG-3') and 3' conserved segment (3'-CS; 5'-AAGCAGACTTGACCTGA-3').6 PCR reactions (total volume 100 µL) contained 10 ng plasmid DNA, 200 µM dNTPs, 2.5 U pfuTurbo DNA polymerase (Stratagene, La Jolla, CA, USA), 10x reaction buffer and 0.3 µM of each primer. One cycle of PCR was run at 95°C for 1 min, followed by 30 cycles each comprising at 95°C for 30 s, 55°C for 30 s and 72°C for 1.5 min. The amplicon(s) were then sequenced and sequence comparisons were made with the BLAST search program.
Nucleotide sequence accession number
The sequence described in this study has been deposited in GenBank under accession no. AF170088.
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Results and discussion |
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An amplicon of c. 1600 bp was obtained by PCR. The nucleotide sequence showed the presence of two genes, the dfr gene and the aadA gene (Figure). Both of these genes were flanked by integrase recombination core sites and had conserved features at the 3' ends of the genes with a 59-base element,1 suggesting that each gene was present as a gene cassette inserted into the variable region between the 5' and 3' conserved segments of a class 1 integron. The 1600 bp amplicon was also ligated with the pGEM-T easy vector (Promega, Madison, WI, USA) and then transformed into E. coli JM101. Transformants containing the amplicon were resistant to trimethoprim, spectinomycin and streptomycin, with MICs of >2048, 128 and 64 mg/L, respectively. This result indicated that dfr and aadA genes encode trimethoprim and spectinomycin/streptomycin resistance, respectively.
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Sixteen different types of plasmid-borne DHFR have been found in Gram-negative bacteria and have been characterized and grouped on the basis of their nucleotide sequences and kinetic properties.7 Family 1, the largest and most prevalent of these groups, includes the proteins encoded by dfrI, dfrIb, dfrV, dfrVI, dfrVII and dfrXV. The genes encoding these trimethoprim-resistant DHFRs have been found as gene cassettes inserted in integrons.9 The DHFRs in family 1 are 6488% identical and the polypeptides are 157 amino acids long. All proteins of family 1 are highly resistant to trimethoprim (>1000 mg/L). Our study demonstrated that the predicted polypeptide product of dfr17 had 91% identity with DHFR type VII and 6368% identity with other DHFRs in family 1. dfr17 also conferred a high level of resistance to trimethoprim. Thus, the product of the dfr17 gene appears to be a new cassette-encoded DHFR belonging to family 1.
The second cassette, spanning 895 nucleotides and extending from base 618 to base 1512, contained an ORF of 789 nucleotides starting at an ATG codon. This coding sequence, designated aadA4, shared 94% identity with the aadA3 gene (GenBank accession no. Z50802). However, the coding region of the aadA4 gene was not highly homologous to those of other known streptomycin/spectinomycin resistance genes, such as aadA1 and aadA2 (approximately 57% and 56% homology, respectively). The aadA4 59-base element was 57 bp long and the sequence shared approximately 70% identity with those of the 59-base elements associated with aadA1 and aadA2. Therefore, aadA4 may, like aadA1 and aadA2, be a cassette-borne aminoglycoside adenylyltransferase gene.
Most gene cassettes are usually inserted in the same orientation and are under the control of the common promoter Pant, located in the 5' conserved segment.1 Sequence analysis of the 5' conserved segment (intI1) of the integron in E. coli EC107 revealed a Pant promoter consisting of 35 (TGGACA) and 10 (TAAACT) motifs separated by 17 nucleotides. This promoter configuration has been identified as a hybrid promoter with intermediate strength.10 The secondary promoter P2 was also found downstream of Pant and consisted of the sequence 35TTGTTA-[N14]-10TACAGT. However, P2 is probably inactive because of the short spacing (14 nucleotides) between the 35 and 10 hexamers.10
In this paper we report two new gene cassettes, dfr17 (encoding trimethoprim resistance) and aadA4 (encoding spectinomycin/streptomycin resistance), inserted in an E. coli class 1 integron. Different types of cassette-encoded DHFRs and AADs exist. The integron carrying the dfr17 and aadA4 cassettes was located on a conjugative plasmid, pEC1072. The transferable plasmid could contribute to the horizontal spread of integron-associated antibiotic-resistant gene cassettes. Thus, dissemination of dfr17 and aadA4 cassettes among clinical isolates should be traced. Whether dfr17 and aadA4 are found in gene cassettes apart from each other also needs to be studied further.
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Acknowledgments |
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Notes |
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Present address. Department of Food Sanitation, Tajen Institute of Technology, Ping Tung 907, Taiwan, Republic of China.
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References |
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2 . National Committee for Clinical Laboratory Standards. (1993). Performance Standards for Antimicrobial Disk Susceptibility Test: Approved Standard M2-A5. NCCLS, Villanova, PA.
3 . National Committee for Clinical Laboratory Standards. (1993). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A3. NCCLS, Wayne, PA.
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Huovinen, P., Sundström, L., Swedberg, G. & Sköld, O. (1995). Trimethoprim and sulfonamide resistance. Antimicrobial Agents and Chemotherapy 39, 27989.
8 . Burnside, J. M. & Groot Obbink, D. J. (1996). Plasmid pDGO100 contains a second integron with the trimethoprim resistance gene dfrA7 as the inserted cassette. Plasmid 35, 6770.[ISI][Medline]
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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.
10 . Lévesque, C., Brassard, S., Lapointe, J. & Roy, P. H. (1994). Diversity and relative strength of tandem promoters for the antibiotic-resistance genes of several integrons. Gene 142, 4954.[ISI][Medline]
Received 29 October 1999; returned 10 January 2000; revised 31 January 2000; accepted 15 February 2000