Dihydrofolate Reductase Type Research Articles

Novel Dihydrofolate Reductases Isolated from Epidemic Strains of Trimethoprim/Sulfamethoxazole-Resistant Shigella sonnei

Two strains of trimethoprim-resistant Shigella sonnei bearing R plasmids pBH600 and pBH700 each elaborated a dihydrofolate reductase (DHFR) and were moderately resistant to trimethoprim (minimum inhibitory concentrations, 128 and 256 micrograms/ml, respectively). Neither plasmid hybridized to probes for DHFR types I, II, or III. The trimethoprim resistance genes from the R plasmids resided on a 1600-base pair (bp) PstI fragment of pBH600 and an 1800-bp PstI fragment of pBH700. Isoelectric focusing showed distinct isoelectric points for the enzymes coded for on pBH600 (5.3) and pBH700 (5.6-5.7). Trimethoprim-resistant S. sonnei from 10 locations in nine states were examined. Isolates from 8 locations hybridized only to a pBH700-derived probe and one isolate hybridized to a pBH600-derived probe. These two trimethoprim resistance genes appear novel. The gene on plasmid pBH700 codes for an enzyme that seems widespread among S. sonnei isolates in the USA.

Cloning of the type VII trimethoprim-resistant dihydrofolate reductase gene and identification of a specific DNA probe

A 1.3 kb HinIII fragment encoding the type VII trimethoprim-resistant dihydrofolate reductase gene was cloned into pBR322. Unidirectional deletion of this cloned fragment with exonuclease III identified the start of the dihydrofolate reductase gene. An internal 300bp EcoRV fragment was identified which could be used as a specific non-radioactive DNA probe to distinguish bacteria carrying the type VII gene from those carrying genes encoding other known dihydrofolate reductase types.

Cloning of the type VII trimethoprim-resistant dihydrofolate reductase gene and identification of a specific DNA probe

A 1.3 kb HindIII fragment encoding the type VII trimethoprim-resistant dihydrofolate reductase gene was cloned into pBR322. Unidirectional deletion of this cloned fragment with exonuclease III identified the start of the dihydrofolate reductase gene. An internal 300bp EcoRV fragment was identified which could be used as a specific non-radioactive DNA probe to distinguish bacteria carrying the type VII gene from those carrying genes encoding other known dihydrofolate reductase types.

Dissemination of Trimethoprim-Resistant Clones of Shigella sonnei in Bulgaria

Bacterial dysentery due to Shigella sonnei remains a serious public health problem in developed countries, including Bulgaria. At the National Shigella Reference Laboratory in Sofia, 17,126 strains of S. sonnei from epidemics and sporadic cases collected from 1973 to 1987 were studied. Antibiotic susceptibility testing, phage typing, colicin typing, and biotyping were performed for all strains to allow intraspecies differentiation and to track any clonal distribution. Of all strains, 84.3% were resistant to one or more antimicrobials, the most frequent being tetracycline (Tet), streptomycin (Str), sulfonamide (Sul), chloramphenicol (Chl), ampicillin (Amp), and trimethoprim (Tmp). Resistance patterns most prevalent for successive 5-y periods included Tet, StrSulTet, and AmpChlStrSulTet, respectively. For the final 5 y, a new pattern (AmpKanStrSulTetTm [Kan = kanamycin]) was spread throughout the country by two trimethoprim-resistant clones. High-level resistance to trimethoprim (MIC greater than 1500 micrograms/mL) in both clones was determined by dihydrofolate reductase type I. The genes for trimethoprim resistance were located on a conjugative R-plasmid of approximately 145 kilobases which cotransferred all other antimicrobial resistances. A similar-sized R-plasmid had been found in earlier isolates of Bulgarian S. sonnei, suggesting that new antimicrobial resistance genes had been sequentially added to an ancestral R-plasmid. Controlling the expression of these new as well as older antimicrobial resistances, particularly for enteric pathogens, must involve reduction in usage of generic antimicrobial agents.

High-level resistance to trimethoprim in Shigella sonnei associated with plasmid-encoded dihydrofolate reductase type I.

By DNA hybridization, the gene encoding dihydrofolate reductase type I was found in 58 of 59 highly trimethoprim-resistant clinical isolates of Shigella sonnei obtained from 1981 through 1987 in Madrid, Spain. No strain harbored the type II gene. In selected strains, the type I gene was demonstrated to be in a plasmid.

Biotinylated DNA probes for trimethoprim-resistant dihydrofolate reductases types IV and V.

DNA probes for trimethoprim-resistant dihydrofolate reductases types IV and V were constructed and characterized. The type IV probe consisted of a 1.7 kb ClaI fragment of recombinant plasmid pUK1148, while the type V probe was a 0.5 kb HincII fragment of plasmid pLK09. Each probe was biotinylated and tested in non-isotopic hybridization experiments with control plasmids carrying genes for known dihydrofolate reductase types. Both probes were highly specific and will therefore be useful in epidemiological studies for monitoring the spread of these unusual plasmid-encoded enzymes.

Characterization of a staphylococcal trimethoprim resistance gene and its product.

Resistance to trimethoprim (Tp) is mediated by a plasmid-encoded gene in staphylococci. The gene is responsible for high-level resistance (MIC, greater than 1,000 micrograms/ml) in both its native host and when cloned on high-copy-number vectors in Escherichia coli. Analysis of subclones of the staphylococcal Tp gene on E. coli expression vectors and estimation of the size of full and truncated proteins produced in E. coli minicells generated an approximate size limit of 505 base pairs for the gene and 18,500 daltons for the gene product. Crude extracts of E. coli containing the cloned gene had dihydrofolate reductase (DHFR) specific activity that was more than 100 times greater than that of control cells and more than 1,000 times more resistant to trimethoprim inhibition. The amount of trimethoprim required for a 50% reduction in the specific activity of staphylococcal DHFR differed from those of cells containing DHFR types I, II, or III, enzymes mediating Tp in members of the family Enterobacteriaceae. In addition, the size of the monomeric staphylococcal DHFR protein was larger than that of any of the gram-negative DHFRs both compared with published sequence data and as observed by direct comparison on polyacrylamide gels. Finally, there was no homology between a DNA fragment containing the cloned staphylococcal gene and DNA encoding any of the gram-negative DHFRs. Thus, the staphylococcal Tp gene codes for a single protein with DHFR activity that appears to be unrelated to DHFR genes that mediate Tp in members of the Enterobacteriaceae.

Rapid detection of a specific trimethoprim resistance gene using a biotinylated DNA probe.

A DNA probe specific for the dihydrofolate reductase (DHFR) type I gene was labelled with biotin by the process of nick-translation and used to screen 83 independently-derived trimethoprim R plasmids from Enterobacteriaceae. Hybridization was detected using streptavidine and a biotin-conjugated alkaline phosphatase to generate an insoluble coloured precipitate following the addition of an appropriate dye. Sixty-eight plasmids (81.9%) hybridized with the probe for DHFR type I. The method could be adapted for use with any antibiotic resistance gene for which a suitable DNA probe is available and has none of the drawbacks associated with the use of radioactively-labelled DNA in hybridization techniques.

Transposable resistance to trimethoprim and 0/129 in Vibrio cholerae.

Vibrio cholerae biotype el tor strain BM2508, resistant to trimethoprim, 0/129, streptomycin and spectinomycin was isolated from the faeces of a child with severe diarrhoea. Resistance to trimethoprim and 0/129 was due to a dihydrofolate reductase type I and resistance to streptomycin-spectinomycin to a 3'',9-aminoglycoside-aminocyclitol adenylyltransferase. The resistance genes were not transferable to Escherichia coli and, as inferred from ultracentrifugation in cesium chloride-ethidium bromide and agarose gel electrophoresis of crude bacterial lysates, were located on the chromosome. The resistance genes were transposed to multiple sites of plasmids belonging to incompatibility groups 6-C and P, introduced in BM2508 and were subsequently transferred to E. coli (rec-), Salmonella typhimurium, V. cholerae and V. parahaemolyticus strains where they re-transposed into the chromosome. Analysis of plasmid DNA from the transconjugants by agarose gel electrophoresis following digestion with HindIII and by Southern hybridization using a ColEl::Tn7 probe indicated the presence of a 14-kilobase transposon, Tn1527, closely related to Tn7. The emergence of Tn1527 in V. cholerae may lead to prophylactic and therapeutic failures due to trimethoprim resistance and to bacterial misidentification because of cross resistance to 0/129.

Molecular epidemiology of resistance to trimethoprim in enterobacteria isolated in a Parisian hospital

Between January, 1981 and December, 1984, 419 strains of enterobacteria isolated from patients at the Hôpital Saint-Joseph were studied for (1) the level of resistance to trimethoprim (Tp) by determination of minimal inhibitory concentration (MIC), (2) transferability of this resistance by conjugation into Escherichia coli, (3) plasmid content of wild-type strains and transconjugants by agarose gel electrophoresis of crude bacterial lysates and by incompatibility grouping, and (4) type of dihydrofolate reductase (DHFR) by colony hybridization with probes specific for DHFR types I and II. Tp resistance was defined as MIC greater than or equal to 4 micrograms/ml and high-level resistance by MIC greater than or equal to 1000 micrograms/ml. Amongst the strains studied, 90% were resistant to high levels of Tp, while 10% had low-level resistance to Tp was detected in 180 strains corresponding to 185 plasmids. In the vast majority of the plasmids, resistance to Tp was associated with resistance to sulphonamide (94%), streptomycin (75-90%), ampicillin (75-90%) and chloramphenicol (65-80%). Plasmids conferring resistance to Tp were often large, most (84%) ranging in size from 90 to 175 Kb. They belonged to six different incompatibility groups and Inc6-C was the most prevalent (34 to 75%). The study of the distribution of the dfr genes by colony hybridization in 183 transconjugants and 89 strains with non-transferable Tp resistance revealed the presence of dfrI genes in most of these strains (48 and 53%, respectively). DHFR of types I and II were found in only 3% of the transconjugants, but in 15% of the strains with non-transferable resistance. DHFR of other types were found equally (15%) in strains with transferable and non-transferable resistance. The high incidence of the type I enzyme among the Tp-resistant strains probably results from the integration of transposon Tn7 into the chromosome or into a non-transferable plasmid.

Increasing resistance to trimethoprim-sulfamethoxazole among isolates of Escherichia coli in developing countries.

Resistance of Escherichia coli to trimethoprim (TMP)-sulfamethoxazole remains at 3%-8% at many medical centers within the United States. In this study a 44% resistance rate was observed among E. coli isolated at a pediatric hospital in Santiago, Chile, and a 40% resistance rate at a general teaching hospital in Bangkok, Thailand. Most isolates were from urinary tract infections and showed high-level resistance (minimal inhibitory concentration of TMP greater than 1,000 micrograms/ml). Nineteen of 35 isolates tested transferred resistance to TMP; most cotransferred resistance to streptomycin and sulfonamides. Dihydrofolate reductase type I was detected by gene probing in 14 of 35 strains. Subsequent investigations in Brazil, Honduras, and Costa Rica revealed that this high rate of resistance was not an isolated phenomenon.

Tn7-encoded proteins.

Proteins encoded by Tn7 have been studied in Escherichia coli maxicells harbouring either various deleted ColE1::Tn7 plasmids or Tn7 fragments cloned in pBR322. Six Tn7-encoded proteins were detected and named p18, p32, p40, p54, p85-a and p85-b according to their apparent molecular weight. Protein p18 is dihydrofolate reductase type I and p32 is probably the protein conferring resistance to streptomycin/spectinomycin. Both genes map on the left-hand part of Tn7. The genes for the four other proteins are located on the right-hand part of Tn7. We propose that they fully cover a 6.9 kb DNA fragment without any overlapping. Starting from the right-hand end towards the middle of the transposon, these four genes are in the following order: p85-a, p54, p40 and p85-b. Transposition of Tn7 onto E. coli plasmids requires the proteins p85-a, p85-b, p54 and p40. However, transposition onto the chromosome does not require the p85-b and p40 products.