Identification of a cassette-borne dfrA7-like gene that shows a 97 bp extension at the 3'-end of the reading frame

Kayode K. Ojoa,b, Diana E. Waturangia,c, H. Akin Odelolab, Antonius Suwantoc,d and Stefan Schwarza,*

a Institut für Tierzucht und Tierverhalten, Bundesforschungsanstalt für Landwirtschaft (FAL), Dörnbergstrasse 25-27, 29223 Celle, Germany; b Department of Pharmaceutical Microbiology and Clinical Pharmacy, College of Medicine, University of Ibadan, Nigeria; c Department of Biology, Faculty of Science and Mathematics, IUC Biotechnology, Bogor Agricultural University, Bogor; d South East Asian Regional Center for Tropical Biology (SEAMEO-BIOTROP), Bogor, Indonesia

Sir,

Sulphonamides and trimethoprim are among the most frequently used antimicrobial drugs in Nigeria, where the combination of both drugs is used preferentially for the treatment of urinary tract infections. However, sulphonamides and trimethoprim are commonly available, alone or in combination, over the counter in pharmacies in Nigeria without a doctor's prescription. Self-medication and misuse of these drugs favours the development of resistance to sulphonamides and trimethoprim in various bacterial pathogens.

During the course of a study on antimicrobial resistance in uropathogenic Escherichia coli from humans in Nigeria, the multi-resistant E. coli strain UCH10386 was obtained from a 52-year-old female out-patient suffering from a urinary tract infection at the University College Hospital, Ibadan, Nigeria. This strain showed resistance to ampicillin, tetracycline, streptomycin, sulphonamides and trimethoprim. All five resistance properties were associated with a plasmid of c. 58 kb, designated pOJO10386, as confirmed by transformation into the E. coli laboratory strain JM107 and conjugation into the rifampicin-resistant E. coli HK225.

Extended PCR analyses were conducted using previously described primers to determine the type of the resistance genes.1–3 For this, plasmid DNA prepared from an E. coli JM107:pOJO10386 transformant was used. These analyses revealed the presence of the tetracycline-resistance gene tet(A), the two sulphonamide-resistance genes sul1 and sul2, the streptomycin-resistance gene aadA2 and a blaTEM-type ampicillin-resistance gene.

Furthermore, plasmid pOJO10386 was screened for the carriage of class I integrons by PCR using a Pwo polymerase (Peqlab, Erlangen, Germany) with an extended proof-reading activity and primers that corresponded to the 5'-conserved segment (5'-GGCATCCAAGCAGCAAG-3') and the 3'-conserved segment (5'-AAGCAGACTTGACCTGA-3') of class I integrons.3 A single amplicon of c. 0.76 kb was obtained from plasmid pOJO10386, whereas the whole cell DNA of the recipient strain E. coli JM107 did not yield an amplicon. This amplicon was cloned into pCR-BluntII-TOPO (Invitrogen, Groningen, The Netherlands) and the recombinant plasmid was transformed into E. coli TOP10. E. coli TOP10 cells that carried this recombinant plasmid showed resistance to trimethoprim. Sequence analysis of the amplicon conducted on both strands confirmed the presence of a dfrA7-like gene that was part of a gene cassette (FigureGo).4



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Figure. Schematic presentation of the dfrA7 gene cassette from plasmid pOJO10386. The dfrA7 reading frame is shown as an arrow, whereas the 5'- and 3'-conserved segments (5' CS and 3' CS) of the class 1 integron are shown as boxes. The beginning and the end of the cassette are shown in detail below. The translational start (TTG) and stop (TAA) codons are underlined. In the 59-base element, the putative IntI1 integrase binding domains 1L, 2L, 2R and 1R are indicated by arrows. The 7 bp core site of the cassette, which also represents the 1R sequence, is shown in bold type. The positions of the bases absent in the 59-base elements of the dfrA7 cassette from pOJO10386, but present in those of the other dfrA7 cassettes, are shown as lower-case letters marked by arrows. The ‘t’ marked by an asterisk is only present in the sequences deposited under accession nos U31119 and X58425. These two sequences also show the exchange of the double underlined ‘T’ by ‘C’. The sequence of the 764 bp amplicon from pOJO10386 has been submitted to the EMBL database and assigned accession no. AJ419170.

 
The amplicon comprised 764 bp; the 96 bp at the 5'-end corresponded exactly to part of the attI1 site of class I integrons, whereas the 56 bp at the 3'-end were identical to the qacE sequence of the 3'-conserved segment of class I integrons.5,6 The 612 bp between these conserved segments represented a complete dfrA7 gene cassette that differed from known dfrA7 cassettes in several aspects. Homology searches (http://www.ncbi.nlm.nih.gov/BLAST/; last accessed 7 November 2001) revealed three database entries of dfrA7 genes (accession nos AF139109, U31119 and X58425), all of which code for virtually identical proteins of 157 amino acids. In comparison with the other dfrA7 genes, a single bp exchange (a->c in dfrA7 from pOJO10386) at position 178 was found to cause a conservative amino acid exchange at codon 21 from isoleucine in all other DfrA7 variants to leucine in DfrA7 from pOJO10386. However, more important was a mutation at positions 558/559. Instead of the ‘aa’ that was present in all other dfrA7 genes, dfrA7 from pOJO10386 had a single ‘c’ at this position. Although the exchange of ‘a’ by ‘c’ at position 558 did not even change the amino acid (isoleucine) at codon 147, the loss of the second ‘a’ caused a frameshift mutation that extended the dfrA7 reading frame by 97 bp and, consequently, the length of the DfrA7 protein by 32 amino acids. This extension of the reading frame obviously did not reduce the level of trimethoprim resistance mediated by this DfrA7 protein. The original E. coli strain UCH10386, its E. coli JM107:pOJO10386 transformant, its E. coli HK225:pOJO10386 transconjugant and the E. coli TOP10 that carried the dfrA7 amplicon cloned into pCR-BluntII-TOPO exhibited trimethoprim MICs of >256 mg/L, whereas those of the E. coli strains HK225 and TOP10 were <2 mg/L.

Analysis of the 59-base element of the dfrA7 cassette from pOJO10386 (FigureGo) showed that this element consisted of 130 bp, whereas the corresponding elements of the other known dfrA7 genes were 134 bp (accession nos U31119 and X58425) or 133 bp (accession no. AF139109).5 The loss of 4 or 3 bp, respectively, had no influence on the overall symmetry of this element and also did not affect the putative IntI1 integrase binding domains 1L, 2L, 2R and 1R.6 Although in all other dfrA genes the coding sequence ended in the region of the inverse core site of the 59-base element, the dfrA7 reading frame from pOJO10386 ended at a translational stop codon in the final part of the 59-base element (FigureGo). Whether this extension of the reading frame into the 59-base element might influence the mobility of this gene cassette is unknown.

Notes

* Corresponding author. Tel: +49-5141-384673; Fax: +49-5141-381849; E-mail: stefan.schwarz{at}fal.de Back

References

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3 . Sandvang, D., Aarestrup, F. M. & Jensen, L. B. (1997). Characterisation of integrons and antibiotic resistance genes in Danish multiresistant Salmonella enterica Typhimurium DT104. FEMS Microbiology Letters 157, 177–81.[ISI][Medline]

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6 . Stokes, H. W., O'Gorman, D. B., Recchia, G. D., Parsekhian, M. & Hall, R. M. (1997). Structure and function of 59-base element recombination sites associated with mobile gene cassettes. Molecular Microbiology 26, 731–45.[ISI][Medline]