New integron-independent trimethoprim resistance gene dfr24, detected in urinary isolate of Escherichiacoli
Abstract number: p940
Grape M., Sundström L., Kronvall G.
In a material of 69 trimethoprim resistant Gram-negative urinary isolates, as many as 11 resulted negative with PCR for all known classes of integrons and four of the five known dfr-genes not carried by integrons. The mechanisms for trimethoprim resistance in these 11 isolates are the subject for further investigations. Here we report the finding of a previously unknown trimethoprim resistance gene in one of the 11 selected isolates.
A shotgun cloning approach has been used to identify DNA fragments containing the trimethoprim resistance mechanism. The entire DNA from the isolate was digested with four restriction endonucleases separately and randomly ligated into pUC18 vectors. The ligation reactions were transformed into TOP10 E. coli recipient cells without any further purification. The transformed E. coli were grown on antibiotic plates containing trimethoprim and ampicillin to select for vectors with inserted fragments containing the desired resistance mechanism. The fragments were subjected to sequence analysis from primers directed to the flanking regions of the polylinker in the vector.
So far, one isolate not yet characterized with respect to molecular cause of trimethoprim resistance was showed to carry a previously unknown dfr-gene. This gene, which has been named dfr24, is in contrast to most known dfr-genes not carried as a gene cassette in an integron. The gene product, the dihydrofolate reductase DHFR24, is only 38% identical to DHFR8 and 32% identical to DHFR9 which are also encoded by genes not inserted as gene cassettes in integrons. The protein seems however somewhat more related to housekeeping dihydrofolate reductase genes of various organisms. The sequence of the new dfr24 gene has been published at EMBL with accession number AJ972619.
The total number of trimethoprim resistance dfr-genes is probably far from known yet. Of the 25 dfr-genes known at present, 12 were published the last 5 years and the majority of them were identified in sequence analysis of integrons. The new gene detected in this study, dfr24, is only distantly related to integron-borne dfr-gene cassettes, and with this approach of shotgun cloning probably more integron-independent dfr-genes can be identified. Furthermore, the sub-selection of integron-negative trimethoprim-resistant isolates this new gene origins from has a potential for more such discoveries.
|Session name:||XXIst ISTH Congress|
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