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.13 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 (Figure).4
|
Analysis of the 59-base element of the dfrA7 cassette from pOJO10386 (Figure) 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 (Figure
). 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
References
1 . Frech, G. & Schwarz, S. (1999). Plasmid-encoded tetracycline resistance in Salmonella enterica subsp. enterica serovars Choleraesuis and Typhimurium: identification of complete and truncated Tn1721 elements. FEMS Microbiology Letters 176, 97103.[ISI][Medline]
2
.
Ng, L.-K., Mulvey, M. R., Martin, I., Peters, G. A. & Johnson, W. (1999). Genetic characterization of antimicrobial resistance in Canadian isolates of Salmonella serovar Typhimurium DT104. Antimicrobial Agents and Chemotherapy 43, 301821.
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, 17781.[ISI][Medline]
4
.
Huovinen, P., Sundström, L., Swedberg, G. & Sköld, O. (1995). Trimethoprim and sulfonamide resistance. Antimicrobial Agents and Chemotherapy 39, 27989.
5 . Recchia, G. D. & Hall, R. M. (1995). Gene cassettes: a new class of mobile element. Microbiology 141, 301527.[ISI][Medline]
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, 73145.[ISI][Medline]