JAC Advance Access originally published online on September 15, 2006
Journal of Antimicrobial Chemotherapy 2006 58(5):1044-1047; doi:10.1093/jac/dkl366
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dfrA25, a novel trimethoprim resistance gene from Salmonella Agona isolated from a human urine sample in Brazil
1 WHO Collaborating Centre for Antimicrobial Resistance in Foodborne Pathogens, Danish Institute for Food and Veterinary Research Bulowsvej 27, DK-1790 Copenhagen V, Denmark 2 Oswaldo Cruz Institute, Avenida Brasil 4365 21045-900 Rio de Janeiro, Brazil
*Corresponding author. Tel: +45-72-34-6000; Fax: +45-72-34-6001; E-mail: ya{at}dfvf.dk
Received 28 May 2006; returned 19 July 2006; revised 14 August 2006; accepted 14 August 2006
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Abstract |
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Objectives: To describe a novel trimethoprim resistance gene, designated dfrA25, which was detected as a gene cassette within a class 1 integron in Salmonella Agona.
Methods: The gene was cloned into Escherichia coli MT102 and resistance to 10 different antimicrobial drugs was measured. A phylogenetic tree was constructed based on representative trimethoprim-resistance-mediating DfrA proteins retrieved from GenBank. Filter-mating experiments and Southern blots of plasmid preparations were performed with the donor and selected transconjugants.
Results and conclusions: dfrA25 encodes a dihydrofolate reductase of 157 amino acids with closest identity (85%) to dfrA5 dihydrofolate reductase. dfrA25 was located on a transferable plasmid (
150 kb) that also harboured the tetracycline resistance gene tet(A).
Keywords: integrons , gene cassettes , dihydrofolate reductase
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionGenBank submissionTransparency declarationsReferences |
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Trimethoprim competitively inhibits the enzyme dihydrofolate reductase, which is responsible for the reduction of dihydrofolate to tetrahydrofolate.1,2 Acquired resistance to trimethoprim includes mutations in the promoter region or in the dihydrofolate reductase structural gene,1,2 but the most common trimethoprim resistance mechanism is the supplementation of a trimethoprim-sensitive dihydrofolate reductase with a trimethoprim-resistance-mediating dihydrofolate reductase, resulting in high-level trimethoprim resistance in various bacteria. Until now more than 30 different trimethoprim-resistance-mediating dihydrofolate reductase (dfr) genes, subdivided into two major types, 1 and 2 (referred to as dfrA and dfrB), have been observed.3,4 A new trimethoprim resistance gene is defined when the dihydrofolate reductase protein (DHFR) encoded by the gene has <95% identity at the amino acid level to known DHFR proteins.2 The association of these genes with mobile genetic elements such as transposons or plasmids has often been observed, and the presence of these genes in cassettes as part of integrons is also common.5,6
In Salmonella various dfr genes have been detected.6 In a previous study, investigating the prevalence of integrons and resistance genes in Salmonella spp. isolates from Brazil, trimethoprim resistance encoded as gene cassettes in class 1 integrons was found in 41% of the isolates and a putative trimethoprim resistance gene cassette was detected within a class 1 integron in Salmonella Agona (no. 32) isolated from human urine.6 In the present study we characterized this novel trimethoprim resistance gene, designated as dfrA25.
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Materials and methods |
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The DNA sequence of the gene cassette containing the putative trimethoprim resistance gene dfrA25 of Salmonella Agona (no. 32) was analysed by use of vector NTI Suite 8. In order to determine whether dfrA25 conferred resistance to trimethoprim, the gene was cloned into Escherichia coli MT102. A PCR product was amplified by use of the primers Salm32Ecor1 (5'-GCCGGAATTCTAACCCAGGACGAGTACC-3') and Salm32BamH1 (5'-CGCGGATCCCCGTCAATTGCATAGCTTTG-3') flanking the gene cassette. The PCR product was digested with EcoRI and BamHI and ligated into the plasmid pLOW1, which was partially digested with the same enzymes.7 The ligation product was electroporated into E. coli MT102 and presumptive clones were selected on Luria–Bertani (LB) agar supplemented with chloramphenicol 20 mg/L. Plasmids were purified from selected colonies using the QIAprep Spin Miniprep Kit (Qiagen, Germany). A 1166 bp fragment was sequenced with the primers M13F (5'-GTAAAACGACGGCCAGT-3') and M13R (5'-GCGGATAACAATTTCACACAGG-3') to make sure that the right gene was inserted. The clone (YA32) was checked for resistance to ten different antimicrobial drugs (ampicillin, chloramphenicol, gentamicin, nalidixic acid, neomycin, spectinomycin, streptomycin, sulfamethoxazole, tetracycline and trimethoprim) by use of a commercially dehydrated panel, Sensititre (Trek Diagnostic Systems, UK), as described by the NCCLS.8
A phylogenetic analysis at the amino acid level was performed by making a phylogenetic tree based on one representative microorganism for each trimethoprim-resistance-mediating DfrA protein. The multiple alignments were made by use of the program ClustalX (version 1.81); an N-J tree was made based on this alignment, which was used as a guide tree for the final alignment used to produce the final bootstrap N-J tree.
Filter-mating experiments with the donor Salmonella Agona (no. 32) to the recipients E. coli 1000R and Salmonella Typhimurium JEO3817 were performed in order to demonstrate horizontal transfer of the class 1 integron with dfrA25 and possible co-transfer of tet(A).9 The mating experiments were performed as described previously by Agersø and Sandvang.9 Transconjugants were selected on LB agar plates supplemented with rifampicillin 50 mg/L and trimethoprim 32 mg/L or tetracycline 16 mg/L, respectively. Presumptive transconjugants were checked by PCR for the presence of class 1 integron [primer pairs: qacE
1-F (5'-ATC GCA ATA GTT GGC GAA GT-3')/qacE1-B (5'-CAA GCT TTT GCC CAT GAA GC-3') targeting qacE1, and Att-1-F (5'-CGG GCA TCC AAG CAG CAA-3')/3'CS-B (5'-CGA TTA TGA CAA CGG CGG AAG GGG C-3') targeting the variable region of the integron] and tet(A) [primer pair: tet(A)-1 (5'-GTAATTCTGAGCACTGTCGC-3')/tet(A)-2 (5'-CTGCCTGGACAACATTGCTT-3')] as described previously.9 Plasmids were purified from the donor, transconjugants and recipients by use of the NucleoBond PC100 plasmid preparation kit (Macherey & Nagel, Germany) and electrophoresed (2 V/cm) for 6 h on a 0.8% agarose gel. The blots were hybridized with two digoxigenin-labelled DNA probes for the presence of the 3' segment of class 1 integrons using the PCR product (225 bp) amplified by the primers qacE1-F and qacE1-B and for tet(A) (956 bp) using the PCR products amplified by tet(A)-1 and tet(A)-2, respectively.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionGenBank submissionTransparency declarationsReferences |
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This gene cassette contained a 471 bp ORF with 85% identity at the amino acid level to the trimethoprim-resistant dihydrofolate reductase DfrA5 (GenBank accession no. DQ267940 [GenBank] ). Downstream of the dihydrofolate reductase gene a putative 59 bp element (nt 507–593) was found with 92% identity to the corresponding sequence of a putative 59 bp element found downstream of a dfrA14 gene cassette in E. coli (Z50805 [GenBank] ) and on the plasmid pRSB107 (AJ851089 [GenBank] ). No other gene cassettes were observed within this class 1 integron.
The clone (YA32) that contained the dfrA25 gene ligated into pLOW1 conferred resistance to chloramphenicol (marker for the plasmid pLOW1), streptomycin (chromosomal) and trimethoprim (MIC > 32 mg/L), which confirmed that DfrA25 conferred resistance to trimethoprim. The phylogenetic tree confirmed DfrA25 to be most closely related to DfrA5 (Figure 1). DfrA25 clustered together with DfrA5 and DfrA14, both DHFR proteins found in Salmonella and E. coli.10–12 DfrA5 has also been found in Shigella13 and Vibrio cholerae.14
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Based on the trimethoprim resistance phenotype and the phylogenetic relationship of the protein with other Dfr type A proteins, we predict this gene to encode a novel trimethoprim-resistance-mediating dihydrofolate reductase named dfrA25 according to the guidelines for naming new trimethoprim resistance genes.2
dfrA25 was found in one isolate of Salmonella Agona isolated from a human urine sample in Brazil. This isolate was also resistant to sulfamethoxazole, encoded by sul1 (on class 1 integron), and to tetracycline, encoded by tet(A). The ability of these resistance genes to spread horizontally between Salmonella and other Gram-negative bacteria was investigated. Horizontal transfer was observed to both recipients (Table 1). The transfer frequency was more than 1000-fold higher to the E. coli recipient than to the Salmonella recipient, so even though horizontal transfer to both recipients was observed, transfer seemed to depend very much on the choice of recipient. All transconjugants were positive for both class 1 integron and tet(A). The donor and transconjugants contained a 150 kb plasmid. The Southern blot showed class 1 integron and tet(A) to be present on the 150 kb plasmid in both donor and transconjugants. Large horizontally transferable plasmids with tet(A) and class 1 integrons have been observed in Salmonella and other species as well.15 How related these large plasmids are is not well studied, but this novel trimethoprim resistance gene dfrA25 has a great potential to be spread among Salmonella and other Gram-negative species along with both tetracycline and sulphonamide resistance.
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GenBank submission |
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The sequence of 593 bp was submitted to GenBank (accession no. DQ267940 [GenBank] ).
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Transparency declarations |
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None to declare.
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Acknowledgements |
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We would like to thank Berith Kummerfeldt and Jane Larsen for excellent technical assistance and the WHO Global Salmonella Survey Program for financial support.
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References |
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1 Huovinen P, Sundstrom L, Swedberg G, et al. (1995) Trimethoprim and sulfonamide resistance. Antimicrob Agents Chemother 39:279–89.
2 Sköld O. (2001) Resistance to trimethoprim and sulfonamides. Vet Res 32:261–73.[CrossRef][ISI][Medline]
3
Pattishall KH, Acar J, Burchall JJ, et al. (1977) Two distinct types of trimethoprim-resistant dihydrofolate reductase specified by R-plasmids of different compatibility groups. J Biol Chem 252:2319–23.
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White PA and Rawlinson WD. (2001) Current status of the aadA and dfr gene cassette families. J Antimicrob Chemother 47:495–6.
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Lee JC, Oh JY, Cho JW, et al. (2001) The prevalence of trimethoprim-resistance-conferring dihydrofolate reductase genes in urinary isolates of Escherichia coli in Korea. J Antimicrob Chemother 47:599–604.
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Peirano G, Agersø Y, Aarestrup FM, et al. (2006) Occurrence of integrons and antimicrobial resistance genes among Salmonella enterica in Brazil. J Antimicrob. Chemother 58:305–9.
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8 National Committee for Clinical Laboratory Standards. (2003) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Sixth Edition: Approved Standard M7-A6NCCLS, Villanova, PA, USA.
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Agersø Y and Sandvang D. (2005) Class 1 integrons and tetracycline resistance genes in Alcaligenes, Arthrobacter, and Pseudomonas spp. isolated from pigsties and manured soil. Appl Environ Microbiol 71:7941–7.
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Young HK, Oumsieh MJ, McIntosh ML. (1994) Nucleotide sequence and genetic analysis of the type Ib trimethoprim-resistant, Tn4132-encoded dihydrofolate reductase. J Antimicrob Chemother 34:715–25.
11 Sundstrom L, Radstrom P, Swedberg G, et al. (1988) Site-specific recombination promotes linkage between trimethoprim- and sulfonamide resistance genes. Sequence characterization of dhfrV and sulI and a recombination active locus of Tn21. Mol Gen Genet 213:191–201.[CrossRef][ISI][Medline]
12
Ojo KK, Kehrenberg C, Schwarz S, et al. (2002) Identification of a complete dfrA14 gene cassette integrated at a secondary site in a resistance plasmid of uropathogenic Escherichia coli from Nigeria. Antimicrob Agents Chemother 46:2054–5.
13 Navia MM, Ruiz J, Vila J. (2004) Molecular characterization of the integrons in Shigella strains isolated from patients with traveller's diarrhea. Diagn Microbiol Infect Dis 48:175–9.[CrossRef][ISI][Medline]
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Thungapathra M, Amita M, Sinha KK, et al. (2002) Occurrence of antibiotic resistance gene cassettes aac(6')-Ib, dfrA5, dfrA12, and ereA2 in class 1 integrons in non-O1, non-O139 Vibrio cholerae strains in India. Antimicrob Agents Chemother 46:2948–55.
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Guerra B, Soto S, Helmuth R, et al. (2002) Characterization of a self-transferable plasmid from Salmonella enterica serotype Typhimurium clinical isolates carrying two integron-borne gene cassettes together with virulence and drug resistance genes. Antimicrob Agents Chemother 46:2977–81.
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