Isolation and characterization of dihydrofolate reductase from trimethoprim-susceptible and trimethoprim-resistant Pseudomonas cepacia

Abstract

Trimethoprim resistance was investigated in cystic fibrosis isolates of Pseudomonas cepacia. Determination of the MIC of trimethoprim for 111 strains revealed at least two populations of resistant organisms, suggesting the presence of more than one mechanism of resistance. Investigation of the antibiotic target, dihydrofolate reductase, was undertaken in both a susceptible strain and a strain with high-level resistance (MIC, >1,000 ,ug/ml). The enzyme was purified by using ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. Specific activities, molecular we;ghts, isoelectric points, and substrate kinetics were similar for both enzymes. However, the dihydrofolate reductase from the trimethoprim-resistant strain demonstrated decreased susceptibility to inhibition by trimethoprim and increased susceptibility to inhibition by methotrexate, suggesting that these two enzymes are not identical. We conclude that the mechanism of trimethoprim resistance in this strain with high-level resistance is production of a trimethoprim-resistant dihydrofolate reductase.Pseudomonas cepacia is an emerging pathogen in children and young adults with cystic fibrosis (CF (24). In a subpopulation of female adolescents with advanced pulmonary disease, severe necrotizing pneumonia and death have occurred (21).An important problem in the treatment of infections caused by P. cepacia is the high incidence of antibiotic resistance. CF isolates, in particular, are often resistant to the P-lactam agents and aminoglycosides used to treat infections caused by Pseudomonas aeruginosa (4, 7, 11). Trimethoprim is an agent which has demonstrated both in vitro and in vivo efficacy against P. cepacia, but up to 60% of isolates from CF patients are resistant to trimethoprim (14). Resistance mechanisms which have been described for trimethoprim include decreased permeability, synthesis of a trimethoprim-resistant dihydrofolate reductase (DHFR), or overproduction of the constitutive, trimethoprim-susceptible enzyme (13). This study describes the isolation and characterization of DHFR from trimethoprim-susceptible and trimethoprim-resistant strains of P. cepacia. MATERIALS AND METHODS Materials

Antifolate Activity of Epigallocatechin Gallate against Stenotrophomonas maltophilia

“…A comparison of the kinetic properties of the DHFRs isolated from the two strains is shown in Table 2. The calculated K m and k cat values at pH 7.4 were essentially the same for the enzymes from the two isolates and similar to those of DHFRs from other species (3,40). S. maltophilia DHFR was strongly inhibited by MTX (inhibition constant [K i ], 18 pM for isolate 1), showing slow-binding inhibition (12) as described for the inhibition of DHFRs from other biological sources by this compound (38).…”

Therapeutic options for Burkholderia cepacia infections beyond co-trimoxazole: a systematic review of the clinical evidence

“…Production of ␤-lactamases confers resistance to ␤-lactams [91], whilst a substantial outer membrane hydrophilic permeability barrier protects the bacillus from all hydrophilic antimicrobial agents [92]. Other mechanisms reported in the literature include production of modifying enzymes other than ␤-lactamases and alteration of antibiotic targets [91,93]. Apart from intrinsic mechanisms, acquired resistance has also been observed against commonly used antibiotics for infections caused by BCC and other common pathogens in CF patients.…”

Antibiotic resistance in Burkholderia species

“…Purification of the enzyme from trimethoprim susceptible and trimethoprim resistant B. cepacia strains indicated that the protein from the resistant strain was indeed refractory to trimethoprim inhibition (Burns et al, 1989). While this finding is consistent with target alteration, the molecular and genetic basis for this reduced inhibition was not established.…”