Two new gene cassettes, dfr17 (for trimethoprim resistance) and aadA4 (for spectinomycin/streptomycin resistance), inserted in an Escherichia coli class 1 integron

Chung-Yu Chang, Lin-Li Chang, Yu-Hung Chang, Tsong-Ming Lee{dagger}, Yuan-Hung Li and Shui-Feng Chang*

Department of Microbiology, School of Medicine, Kaohsiung Medical University, 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan, Republic of China


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Two new gene cassettes, dfr17 and aadA4, inserted in a class 1 integron of Escherichia coli EC107, are described here. The dfr17 cassette encodes trimethoprim resistance and has 91% identity with the dfrVII dihydrofolate reductase gene. The aadA4 cassette confers resistance to spectinomycin and streptomycin and shows 94% identity with the aadA3 gene. The integron carrying the dfr17 and aadA4 cassettes was located on a conjugative plasmid, pEC1072.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Integrons are specialized genetic elements capable of integrating or mobilizing gene cassettes by site-specific recombination.1 Gene cassettes, most commonly encoding antibiotic resistance, are individual mobile units composed of a gene and an integrase-specific recombination site known as a 59-base element.1 Four types of integron have been identified to date, of which class 1 integrons are the most prevalent among clinical isolates. More than 40 distinct cassettes have been shown to be carried within class 1 integrons.1 In this report, we describe a class 1 integron of Escherichia coli carrying two new gene cassettes, dfr17 and aadA4, which confer resistance to trimethoprim and spectinomycin/streptomycin, respectively.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strain

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 Mueller–Hinton 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.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
E. coli EC107 was resistant to ampicillin, chloramphenicol, gentamicin, spectinomycin, streptomycin, sulphonamides and trimethoprim and carried three plasmids of 148, 119 and 3 kb. The 119 kb plasmid hybridized with the oligonucleotide probe specific for the intI1 gene (data not shown). The conjugation experiment demonstrated that the 119 kb plasmid was transferable. This plasmid was named pEC1072.

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 (FigureGo). 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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Figure. Schematic representation of the integron of E. coli EC107. The line below the integron structure represents the amplicon obtained with primers 5'-CS and 3'-CS. The black box represents the nucleotide sequence deposited in GenBank under accession no. AF170088. Restriction sites shown are E, EcoRV; Ps, PstI; Pv, PvuII. Numbers above the integron structure correspond to sequence positions in GenBank accession no. AF170088.

 
The first gene cassette was 616 bp long and contained an ORF of 471 bp encoding 157 amino acids. The coding sequence, designated dfr17, showed 91% identity with the dfrVII gene found within transposon Tn50867 and in plasmid pDGO100.8 The ORF began with start codon TTG. Despite the atypical start codon, which normally encodes leucine, this was the only codon that was preceded by a plausible Shine–Dalgarno sequence, TAAGGGAGT. Although rare in Gram-negative bacteria, TTG acting as a methionine start codon in E. coli has been reported.9 Furthermore, the use of alternative E. coli start codons GTG and TTG in other dihydrofolate reductases (DHFRs) such as dfrI, dfrV, dfrXV and dfrVII, has been demonstrated previously.9 The dfr17 59-base element was 133 bp long and had 87% sequence homology with the 134 bp 59-base element of the dfrVII cassette. It is known that the members of the family of 59-base elements vary in length. Several groups of closely related 59-base elements have been identified;1 one of these groups includes the longest three 59-base elements, which range in size from 127 to 141 bp and are associated with blaIMP, dfrVII and qacE. The 133 bp 59-base element of dfr17 can be classified within this group.

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 64–88% 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 63–68% 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.


    Acknowledgments
 
This study was supported by grants from the Department of Health, Republic of China (DOH88-TD-1009) and partially from the Kaohsiung Medical University (M8725).


    Notes
 
* Corresponding author. Tel: +886-(7)-312-1101, ext. 2145; Fax: +886-(7)-321-8309; E-mail: shfech{at}cc.kmu.edu.tw Back

{dagger} Present address. Department of Food Sanitation, Tajen Institute of Technology, Ping Tung 907, Taiwan, Republic of China. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Recchia, G. D. & Hall, R. M. (1995). Gene cassettes: a new class of mobile element. Microbiology 141, 3015–27.[ISI][Medline]

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.

4 . Kado, C. I. & Liu, S. T. (1981). Rapid procedure for detection and isolation of large and small plasmids. Journal of Bacteriology 145, 1365–73.[ISI][Medline]

5 . Schwocho, L. R., Schaffner, C. P., Miller, G. H., Hare, R. S. & Shaw, K. J. (1995). Cloning and characterization of a 3-N-aminoglycoside acetyltransferase gene, aac(3)-Ib, from Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 39, 1790–6.[Abstract]

6 . Lévesque, C., Piché, L., Larose, C. & Roy, P. H. (1995). PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrobial Agents and Chemotherapy 39, 185–91.[Abstract]

7 . Huovinen, P., Sundström, L., Swedberg, G. & Sköld, O. (1995). Trimethoprim and sulfonamide resistance. Antimicrobial Agents and Chemotherapy 39, 279–89.[Free Full Text]

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, 67–70.[ISI][Medline]

9 . 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, 2221–4.[Abstract/Free Full Text]

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, 49–54.[ISI][Medline]

Received 29 October 1999; returned 10 January 2000; revised 31 January 2000; accepted 15 February 2000