To Trimethoprim Research Articles

Methionyl-tRNA formyltransferase utilizes 10-formyldihydrofolate as an alternative substrate and impacts antifolate drug action.

Methionyl-tRNA formyltransferase (Fmt)-mediated formylation of Met-tRNAfMet to fMet-tRNAfMet is crucial for efficient initiation of translation in bacteria and the eukaryotic organelles. Folate dehydrogenase-cyclohydrolase (FolD), a bifunctional enzyme, carries out conversion of 5,10-methylene tetrahydrofolate (5,10-CH2-THF) to 10-formyl-THF (10-CHO-THF), a metabolite utilized by Fmt as a formyl group donor. In this study, using in vivo and in vitro approaches, we show that 10-CHO-DHF may also be utilized by Fmt as an alternative substrate (formyl group donor) to formylate Met-tRNAfMet. Dihydrofolate (DHF) formed as a by-product in the in vitro assay was verified by LC-MS/MS analysis. FolD-deficient mutants and Fmt over-expressing strains were more sensitive to trimethoprim (TMP) than the ∆fmt strain, suggesting that the domino effect of TMP leads to inhibition of protein synthesis and strain growth. Antifolate treatment to Escherichia coli showed a decrease in the reduced folate species (THF, 5,10-CH2-THF, 5-CH3-THF, 5,10-CH+-THF and 5-CHO-THF) and increase in the oxidized folate species (folic acid and DHF). In cells, 10-CHO-DHF and 10-CHO-folic acid were enriched in the stationary phase. This suggests that 10-CHO-DHF is a bioactive metabolite in the folate pathway for generating other folate intermediates and fMet-tRNAfMet.

Isolation and Identification of Pathogenic Bacteria Causing Otitis Media in Misan Governorate

This study examined 150 ear swab samples from patients with otitis media who consulted at Al-Sadr Teaching Hospital from January to April 2021 in Misan, Iraq. The participants were aged 14–50 years, among which the infection rate was highest in participants aged 14–22 years and lowest in those aged 40–50 years. Subsequently, bacterial isolates were identified based on their morphology in various culture media and using biochemical tests. Six bacterial species were identified, namely Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus epidermidis. Staphylococcus aureus had the highest infection rate (30%), whereas Staphylococcus epidermidis had the lowest infection rate (8.55%). When the sensitivity of each isolate to antibiotics was determined, Escherichia coli was the most sensitive to trimethoprim (TMP), whereas Pseudomonas aeruginosa was the most resistant to 75% of the tested antibiotics.

The Bacterial Genomic Context of Highly Trimethoprim-Resistant DfrB Dihydrofolate Reductases Highlights an Emerging Threat to Public Health.

Type B dihydrofolate reductase (dfrb) genes were identified following the introduction of trimethoprim in the 1960s. Although they intrinsically confer resistance to trimethoprim (TMP) that is orders of magnitude greater than through other mechanisms, the distribution and prevalence of these short (237 bp) genes is unknown. Indeed, this knowledge has been hampered by systematic biases in search methodologies. Here, we investigate the genomic context of dfrbs to gain information on their current distribution in bacterial genomes. Upon searching publicly available databases, we identified 61 sequences containing dfrbs within an analyzable genomic context. The majority (70%) of those sequences also harbor virulence genes and 97% of the dfrbs are found near a mobile genetic element, representing a potential risk for antibiotic resistance genes. We further identified and confirmed the TMP-resistant phenotype of two new members of the family, dfrb10 and dfrb11. Dfrbs are found both in Betaproteobacteria and Gammaproteobacteria, a majority (59%) being in Pseudomonas aeruginosa. Previously labelled as strictly plasmid-borne, we found 69% of dfrbs in the chromosome of pathogenic bacteria. Our results demonstrate that the intrinsically TMP-resistant dfrbs are a potential emerging threat to public health and justify closer surveillance of these genes.

Gain- and Loss-of-Function Screens Coupled to Next-Generation Sequencing for Antibiotic Mode of Action and Resistance Studies in Streptococcus pneumoniae.

Two whole-genome screening approaches are described for studying the mode of action and the mechanisms of resistance to trimethoprim (TMP) in the Gram-positive Streptococcus pneumoniae The gain-of-function approach (Int-Seq) relies on a genomic library of DNA fragments integrated into a fucose-inducible cassette. The second approach, leading to both gain- and loss-of-function mutation, is based on chemical mutagenesis coupled to next-generation sequencing (Mut-Seq). Both approaches pointed at the drug target dihydrofolate reductase (DHFR) as a major resistance mechanism to TMP. Resistance was achieved by dhfr overexpression either through the addition of fucose (Int-Seq) or by mutations upstream of the gene (Mut-Seq). Three types of mutations increased expression by disrupting a predicted Rho-independent terminator upstream of dhfr Known and novel DHFR mutations were also detected by Mut-Seq, and these were functionally validated for TMP resistance. The two approaches also suggested that an increase in the metabolic flux from purine synthesis to GTP and then to folate can modulate the susceptibility to TMP. Finally, we provide evidence for a novel role of the ABC transporter PatAB in TMP susceptibility. Our genomic screens highlighted novel aspects on the mode of action and mechanisms of resistance to antibiotics.

Antibiotic-Resistant E. coli in Uncomplicated Community-Acquired Urinary Tract Infection.

Routine urine culture testing is not recommended for uncomplicated urinary tract infections (UTIs). As a result, the antibiotic resistance patterns or the organisms causing UTIs are not adequately reflected in routine data. We studied the sensitivity of Escherichia coli (E. coli) to trimethoprim (TMP) and to cotrimoxazole (i.e., trimethoprim/sulfamethoxazole, TMP/SMX) in community-acquired UTI and compared the findings with the resistance data of the Antimicrobial Resistance Surveillance System (ARS). General practitioners and internists in private practice prospectively recruited all of their adult patients with symptoms of a urinary tract infection from May 2015 to February 2016. Urine specimens from all patients were tested (including urine culture testing and antibiotic susceptibility) and infections were defined as uncomplicated or complicated UTIs. 1245 participants from 58 medical practices were enrolled in the study. Pathogenic organisms were found in the urine of 877 patients, of whom 74.5% had E. coli infections. Among the E.-coli-positive UTIs, 52.4% were classified as uncomplicated and 47.6% as complicated. The prevalence of E. coli that was resistant to TMP and to TMP/SMX in uncomplicated UTIs was 15.2% and 13.0%, respectively, compared to 25.3% and 24.4%, respectively, from all UTIs in ARS in 2015. Study participants who had previously taken antibiotics had the highest prevalence of E. coli resistance (30.9%), followed by those who had two or more UTIs within the past six months (28.9%). E. coli with resistance to TMP was significantly less prevalent among the study patients with uncomplicated UTIs than in the routine data of the ARS. Accordingly, TMP should still be considered as an option for the treatment of uncomplicated UTIs. TMP/SMX is considered the agent of second choice because of its side effects. Surveillance systems based on routine data do not yield a representative sample for the evaluation of the resistance situation in patients with uncomplicated UTIs.

Effects of enrofloxacin, ciprofloxacin, and trimethoprim on two generations of Daphnia magna

Multigenerational tests on Daphnia magna were performed exposing two subsequent generation to enrofloxacin (EFX) and its metabolite ciprofloxacin (CPX), and to trimethoprim (TMP). Mortality rate of 100% and 50% was detected in F0 at concentrations of ≥13mgL−1 (EFX) and 50mgL−1 (TMP), respectively. In F1 with respect to F0, both for growth and reproduction, a worsening trend of the response with EFX, a similar response with CPX and an attenuating trend with TMP was observed. Furthermore, the lowest EC20 for reproduction inhibition (1.3mgL−1) was calculated for F1 exposed to EFX. However, other experimentations, longer and more complex, are necessary in order to confirm that EFX is more hazardous to daphnids than CPX and TMP. EC50 measured for the three assayed antibacterials were in the 6.5–37mgL−1 range therefore environmental unrealistic, except in case of exceptional contaminations that may occur in relation to poorly controlled wastewaters from pharmaceutical factories or excessive use of prophylactic treatments in aquaculture.

PS-368 12 Years Data On Resistance Pattern Of Uropathogens: Abstract PS-368 Table 1

Background Urinary tract infection (UTI) is a common bacterial infection in infants and children. Traditionally, Trimethoprim has been the first line antibiotic of choice for the treatment of Coliform UTI. Empirical antibiotic treatment in UTI in children should be based on local surveillance data on the epidemiology and resistance patterns of common uropathogens. This has been endorsed in the guideline on UTI in children by National Institute for Health and Care Excellence in the UK. Aim To evaluate the resistance pattern to commonly used antibiotics in children with culture proven UTI. Methods We retrospectively analysed all cases with proven UTI from 2002–2013. Cases were identified by the clinical coding system and data obtained from the Hospital Information Support System. Results 1151 cases were identified; 95% (1090/1151) had pure growth of E.coli. Microbiology results were analysed to evaluate the sensitivities to commonly used antibiotics. Increasing resistance to Trimethoprim (TMP) and Co-Amoxiclav was observed during 2002–2008. Change in antibiotic policy has resulted in some decline in TMP resistance (Table below) over the recent years. Conclusion This comprehensive data on children with culture proved UTI supports the use of cefalexin as empirical treatment of choice for all presumed UTIs (pending culture results) in our region. Alternatively, TMP can be used (provided that urine sensitivity is known before commencing antibiotic treatment) if it is sensitive to coliform. It is vital to continue to monitor the resistance pattern to ensure early identification of emergence of drug resistance.

A bioorthogonal small-molecule-switch system for controlling protein function in live cells.

Chemically induced dimerization (CID) has proven to be a powerful tool for modulating protein interactions. However, the traditional dimerizer rapamycin has limitations in certain in vivo applications because of its slow reversibility and its affinity for endogenous proteins. Described herein is a bioorthogonal system for rapidly reversible CID. A novel dimerizer with synthetic ligand of FKBP' (SLF') linked to trimethoprim (TMP). The SLF' moiety binds to the F36V mutant of FK506-binding protein (FKBP) and the TMP moiety binds to E. coli dihydrofolate reductase (eDHFR). SLF'-TMP-induced heterodimerization of FKBP(F36V) and eDHFR with a dissociation constant of 0.12 μM. Addition of TMP alone was sufficient to rapidly disrupt this heterodimerization. Two examples are presented to demonstrate that this system is an invaluable tool, which can be widely used to rapidly and reversibly control protein function in vivo.

A Bioorthogonal Small‐Molecule‐Switch System for Controlling Protein Function in Live Cells

AbstractChemically induced dimerization (CID) has proven to be a powerful tool for modulating protein interactions. However, the traditional dimerizer rapamycin has limitations in certain in vivo applications because of its slow reversibility and its affinity for endogenous proteins. Described herein is a bioorthogonal system for rapidly reversible CID. A novel dimerizer with synthetic ligand of FKBP′ (SLF′) linked to trimethoprim (TMP). The SLF′ moiety binds to the F36V mutant of FK506‐binding protein (FKBP) and the TMP moiety binds to E. coli dihydrofolate reductase (eDHFR). SLF′‐TMP‐induced heterodimerization of FKBP(F36V) and eDHFR with a dissociation constant of 0.12 μM. Addition of TMP alone was sufficient to rapidly disrupt this heterodimerization. Two examples are presented to demonstrate that this system is an invaluable tool, which can be widely used to rapidly and reversibly control protein function in vivo.

Prospective Screening of Novel Antibacterial Inhibitors of Dihydrofolate Reductase for Mutational Resistance

Resistance to trimethoprim (TMP) resulting from point mutations in the enzyme drug target dihydrofolate reductase (DHFR) drives the development of new antifolate inhibitors effective against methicillin-resistant Staphylococcus aureus (MRSA). For the past several years we have used structure-based design to create propargyl-linked antifolates that are highly potent antibacterial agents. In order to focus priority on the development of lead compounds with a low propensity to induce resistance, we prospectively evaluated resistance profiles for two of these inhibitors in an MRSA strain. By selection with the lead inhibitors, we generated resistant strains that contain single point mutations F98Y and H30N associated with TMP resistance and one novel mutation, F98I, in DHFR. Encouragingly, the pyridyl propargyl-linked inhibitor selects mutants at low frequency (6.85 × 10(-10) to 1.65 × 10(-9)) and maintains a low MIC (2.5 μg/ml) and a low mutant prevention concentration (1.25 μg/ml), strongly supporting its position as a lead compound. Results from this prospective screening method inform the continued design of antifolates effective against mutations at the Phe 98 position. Furthermore, the method can be used broadly to incorporate ideas for overcoming resistance early in the development process.

Novel LTCC-potentiometric microfluidic device for biparametric analysis of organic compounds carrying plastic antibodies as ionophores: Application to sulfamethoxazole and trimethoprim

Monitoring organic environmental contaminants is of crucial importance to ensure public health. This requires simple, portable and robust devices to carry out on-site analysis. For this purpose, a low-temperature co-fired ceramics (LTCC) microfluidic potentiometric device (LTCC/μPOT) was developed for the first time for an organic compound: sulfamethoxazole (SMX). Sensory materials relied on newly designed plastic antibodies. Sol–gel, self-assembling monolayer and molecular-imprinting techniques were merged for this purpose. Silica beads were amine-modified and linked to SMX via glutaraldehyde modification. Condensation polymerization was conducted around SMX to fill the vacant spaces. SMX was removed after, leaving behind imprinted sites of complementary shape. The obtained particles were used as ionophores in plasticized PVC membranes. The most suitable membrane composition was selected in steady-state assays. Its suitability to flow analysis was verified in flow-injection studies with regular tubular electrodes. The LTCC/μPOT device integrated a bidimensional mixer, an embedded reference electrode based on Ag/AgCl and an Ag-based contact screen-printed under a micromachined cavity of 600 μm depth. The sensing membranes were deposited over this contact and acted as indicating electrodes. Under optimum conditions, the SMX sensor displayed slopes of about −58.7 mV/decade in a range from 12.7 to 250 μg/mL, providing a detection limit of 3.85 μg/mL and a sampling throughput of 36 samples/h with a reagent consumption of 3.3 mL per sample. The system was adjusted later to multiple analyte detection by including a second potentiometric cell on the LTCC/μPOT device. No additional reference electrode was required. This concept was applied to Trimethoprim (TMP), always administered concomitantly with sulphonamide drugs, and tested in fish-farming waters. The biparametric microanalyzer displayed Nernstian behaviour, with average slopes −54.7 (SMX) and +57.8 (TMP) mV/decade. To demonstrate the microanalyzer capabilities for real applications, it was successfully applied to single and simultaneous determination of SMX and TMP in aquaculture waters.

Isolation and Identification of bacterial isolates from ear infection and their sensitivity to usual antibiotics in human and dogs

Bacterial examination was done on 50 dogs suffering from ear infection. Dogs examined include both sexes and their ages were between 5 months to 5 years, In the other hand 35 ear smears from male and female human in ages between 7-40 years were also taken . Isolation and identification of bacterial isolates were done to determine species of zoonotic bacteria in human and dogs, also study of antibiotics sensitivity to these isolates. We identified 137 bacterial isolates from dogs ear belonge to 9 bacterial spp. which are : S. aureus( 27.7%) , Klebsiella spp. ( 13.8%) , E. coli ( 13.1%) , Streptococcus spp. ( 12.4%) ,P. haemolytica ( 8.2%) , P. vulgaris ( 7.2%) , S. epidermidis ( 6.5%) , Salmonella spp. ( 5.8%) , P. mirabilis ( 5.1%) . In human ear smears 95 isolates were identified to 11 bacterial spp. as follows : S. aureus( 26.3%) , Streptococcus spp. ( 15.7%) , S. epidermidis ( 13.6%) , Citrobacter spp. ( 9.4%) , P. mirabilis ( 8.4%) , Klebsiella spp. ( 6.3%) , P. vulgaris ( 5.2%) , Salmonella spp. ( 5.2%) , P. aeruginosa ( 4.2%) , Shigella spp. ( 3.1%) , E. coli ( 2.1%) . Antibiotic sensitivity to dog isolates show high sensitivity to Imipenem (IPM) then Streptomycin ( S) then Rifampin ( RA) then Clindamycin ( DA) and lowest to Erythromycin ( E) respectively . but most isolates were resistant to Cefotaxime( CTX) then Ampicillin ( AM) then Vancomycin ( VA) and lowest to Trimethoprim ( TMP) respectively . The human isolates show high sensitivity to Imipenem ( IPM ) then Streptomycin ( S ) then Erythromycin ( E ) then Rifampin ( RA) then Clindamycin ( DA) finally Vancomycin ( VA) . but most isolates were resistant to Cefotaxime ( CTX ) then Ampicillin ( AM) finally Trimethoprim (TMP). P. aeruginosa was resistant to all antibiotics except Imipenem ( IPM) , Streptomycin ( S) and Erythromycin ( E)

Development of a yeast protein fragment complementation assay (PCA) system using dihydrofolate reductase (DHFR) with specific additives

A yeast protein fragment complementation assay (PCA) system based on dihydrofolate reductase (DHFR) is difficult to be operated because it is not as sensitive to trimethoprim (TMP) as the system using a prokaryotic microorganism. Here, the PCA system using DHFR, specific inhibitors, and a substrate in the yeast Saccharomyces cerevisiae was newly developed. As a model, the human oncoprotein Ras and the Ras-binding domain (RBD) of Raf-1 were individually and genetically fused to DHFR fragment, and each genetic construct was coexpressed under the control of the GAL1 promoter. An interaction between Ras and RBD could be evaluated on the basis of cell proliferation. To establish the experimental conditions for the yeast PCA system based on the DHFR reconstitution, we examined yeast host strains and the concentration of inhibitory additives to prevent endogenous DHFR activity, namely, TMP and sulfanilamide, and the substrate of DHFR, namely, folic acid. The transformant harboring wild-type Ras or its variants showed positive interaction signals, and the order of interactions for combination corresponded to the results of other in vitro assays. Moreover, combinatorial mutated Ras-binding domains were constructed, and the interaction of RBDs with Ras using this yeast PCA system was examined. As a result, various types of mutated clone for RBD were obtained. These demonstrations suggest that the yeast PCA system based on DHFR can be one of good, convenient, and inexpensive tools for investigating eukaryotic protein-protein interactions in vivo.

In Vitro Efficacy of New Antifolates against Trimethoprim-Resistant Bacillus anthracis

Bacillus anthracis is innately resistant to trimethoprim (TMP), a synthetic antifolate that selectively inhibits several bacterial dihydrofolate reductases (DHFRs) but not human DHFR. Previously, we were able to confirm that TMP resistance in B. anthracis (MIC > 2,048 microg/ml) is due to the lack of selectivity of TMP for the B. anthracis DHFR (E. W. Barrow, P. C. Bourne, and W. W. Barrow, Antimicrob. Agents Chemother. 48:4643-4649, 2004). In this investigation, 24 2,4-diaminopyrimidine derivatives, representing a class of compounds with dihydrophthalazine side chains, were screened for their in vitro effects on B. anthracis Sterne and their selectivities for the B. anthracis DHFR. MICs were obtained by a colorimetric (Alamar blue) broth microdilution assay. Purified human recombinant DHFR (rDHFR) and B. anthracis rDHFR were used in a validated enzyme assay to determine the 50% inhibitory concentrations (IC(50)s) and the selectivity ratios of the derivatives. The MICs ranged from 12.8 to 128 microg/ml for all but nine compounds, for which the MICs were > or =128 microg/ml. The IC(50) values for B. anthracis rDHFR ranged from 46 to 600 nM, whereas the IC(50) values for human rDHFR were >16,000 nM. This is the first report on the in vitro inhibitory actions of this class of antifolates against TMP-resistant B. anthracis isolates. The selective inhibition of B. anthracis rDHFR and the in vitro activity against B. anthracis demonstrate that members of this class of compounds have the potential to be developed into clinically important therapeutic choices for the treatment of infections caused by TMP-resistant bacteria, such as B. anthracis.

Functional Cloning of Bacillus anthracis Dihydrofolate Reductase and Confirmation of Natural Resistance to Trimethoprim

Bacillus anthracis is reported to be naturally resistant to trimethoprim (TMP), a drug that inhibits dihydrofolate reductase (DHFR), a key enzyme in the folate pathway. A microdilution broth assay established that the MIC of TMP for B. anthracis Sterne is >2,048 but < or =4,096 microg/ml. A putative DHFR sequence was amplified from B. anthracis Sterne genomic DNA. The PCR product was cloned into the Invitrogen pCRT7/CT-TOPO vector, followed by transformation into Escherichia coli TOP10F' chemically competent cells. Plasmid DNA from a clone showing the correct construct with a thrombin cleavage site attached downstream from the terminus of the cloned PCR product was transformed into E. coli BL21 Star (DE3)pLysS competent cells for expression of the six-histidine-tagged fusion protein and purification on a His-Bind resin column. Functionality of the purified Sterne recombinant DHFR (Sterne rDHFR) was confirmed in an established enzyme assay. The 50% inhibitory concentrations of TMP and methotrexate for the Sterne rDHFR were found to be 77,233 and 12.2 nM, respectively. TMP resistance was observed with E. coli BL21 Star (DE3)pLysS competent cells transformed with the Sterne DHFR gene. Alignment of the amino acid sequence of the Sterne DHFR gene revealed 100% homology with various virulent strains of B. anthracis. These results confirm the natural resistance of B. anthracis to TMP and clarify that the resistance is correlated to a lack of selectivity for the chromosomally encoded gene product. These findings will assist in the development of narrow-spectrum antimicrobial agents for treatment of anthrax.

Iclaprim, a Novel Diaminopyrimidine with Potent Activity on Trimethoprim Sensitive and Resistant Bacteria.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Iclaprim, a novel diaminopyrimidine with potent activity on trimethoprim sensitive and resistant bacteria

Iclaprim, a new selective dihydrofolate inhibitor was synthesized based on rational drug design. Iclaprim's interaction with a resistant Staphylococcus aureus dihydrofolate reductase (DHFR) is outlined in comparison to trimethoprim (TMP). This compound is active against methicillin, TMP and vancomycin resistant strains. Arpida Ltd. is developing Iclaprim for serious hospital infections from Gram-positive pathogens and respiratory tract infections.

Resistance to trimethoprim-sulfamethoxazole.

Sulfonamides have a glorious history. In 1935, they were the first class of true antimicrobial agents with life-saving potency. Today, 66 years later, increased bacterial resistance to sulfonamides and to trimethoprim (TMP), a synthetic antimicrobial agent that is 30 years younger than sulfonamides, has limited their use to only a few indications. In the treatment and prophylaxis of patients with urinary tract infections, trimethoprim-sulfamethoxazole (TMP-SMZ) or TMP alone is still considered the first-line drug of choice, although increased bacterial resistance to these agents has been linked with treatment failure. TMP-SMZ has a possible role as a second- or third-line treatment for patients who have respiratory tract infections. In the developing world, where this inexpensive drug is widely used as first-line treatment, bacterial resistance has caused problems, especially with regard to the treatment of patients with severe respiratory tract infections. Use of TMP-SMZ as prophylaxis for Pneumocystis carinii infection has rapidly increased the multidrug resistance of bacterial pathogens found in human immunodeficiency virus-infected patients. Today, detailed and reliable knowledge on the resistance of bacterial pathogens to both TMP-SMZ and TMP is an essential requirement for the safe and effective use of these drugs in all clinical settings.

Pneumocystis carinii dihydropteroate synthase but not dihydrofolate reductase gene mutations correlate with prior trimethoprim-sulfamethoxazole or dapsone use.

Recent studies of the human Pneumocystis carinii dihydropteroate synthase (DHPS) gene suggest that P. carinii is developing resistance to sulfamethoxazole (SMX) and dapsone. To explore whether P. carinii is also developing resistance to trimethoprim (TMP), the human P. carinii dihydrofolate reductase (DHFR) gene was cloned, DHFR and DHPS genes in 37 P. carinii isolates from 35 patients were sequenced, and the relationship between TMP-SMX or dapsone use and gene mutations was analyzed. The DHFR gene sequences were identical in all isolates except 1 with a synonymous substitution. In contrast, the DHPS gene sequences showed mutations in 16 of the 37 isolates; prior sulfa/sulfone prophylaxis was associated with the presence of these mutations (P<.001). In addition to suggesting that there is less selective pressure on DHFR than on DHPS, this study reinforces the hypothesis that mutations in the DHPS gene are likely involved in the development of sulfa resistance in P. carinii.

Discrimination of a single base change in a ribozyme using the gene for dihydrofolate reductase as a selective marker in Escherichia coli.

For use of ribozymes in vivo, it is desirable to select functional ribozymes in the cellular environment (in the presence of inhibitory factors and limited concentrations of mandatory Mg2+ ions, etc.). As a first step toward this goal, we developed a new screening system for detection in vivo of an active ribozyme from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker. In our DHFR expression vector, the sequence encoding either the active or the inactive ribozyme was connected to the DHFR gene. The plasmid was designed such that, when the ribozyme was active, the rate of production of DHFR was high enough to endow resistance to trimethoprim (TMP). We demonstrated that the active ribozyme did indeed cleave the primary transcript in vivo, whereas the inactive ribozyme had no cleavage activity. Cells that harbored the active-ribozyme-coding plasmid grew faster in the presence of a fixed concentration of TMP than the corresponding cells that harbored the inactive-ribozyme-coding plasmid. Consequently, when cells were transformed by a mixture that consisted of active- and inactive-ribozyme-coding plasmids at a ratio of 1:1, (i) mainly those cells that harbored active ribozymes survived in the presence of TMP and (ii) both active- and inactive-ribozyme-harboring cells grew at an identical rate in the absence of TMP, a demonstration of a positive selection system in vivo. If the background "noise" can be removed completely in the future, the selection system might usefully complement existing selection systems in vitro.