Dihydrofolate Reductases in Some Folate-Requiring Bacteria with Low Trimethoprim Susceptibility

Abstract

Dihydrofolate reductases from the folate-requiring strains Streptococcus faecalis ATCC 8043, Lactobacillus casei ATCC 7496, and Pediococcus cerevisiae ATCC 8081, as well as from Lactobacillus arabinosus, which is not dependent on exogenous folate, were isolated, and their properties were compared to reductases of Escherichia coli B, Staphylococcus aureus, and rat liver reductase. An inhibition profile with six different inhibitors revealed significant differences among all enzymes. All lactobacilli reductases are less sensitive to trimethoprim than the enzymeA of E. coli and S. aureus, the reductase of P. cerevisiae requiring a concentration at least 1,000 times higher for 50% inhibition. Inhibition of growth of S. faecalis by pyrimethanmine and 2,4-diamino-6,7-diisopropyl-pteridine was seen to be much stronger than was predicted from the enzymatic data.Strains such as Streptococcus faecalis, Pediococcus cerevisiae, and Lactobacillus casei are often used for evaluating and characterizing antifolate compounds (e.g., references 9, 10, and 21). They are unable to synthesize their own folate cofactors and require preformed folates or, as in the case of P. cerevisiae, reduced folates for growth. These strains are therefore also used for very sensitive assays of several folate compounds, as they differ in their ability to utilize particular derivatives. They are strongly inhibited by antifolate compounds such as methotrexate or aminopterin, which may be actively transported (14, 27). In P. cerevisiae, the uptake system has been shown to be not identical with that for the transport offolinate (13). The strains are of course resistant to sulfonamides which interfere with the biosynthesis of dihydrofolate at dihydropteroate synthetase.During work on dihydrofolate reductases (EC 1.5.1.3) of different bacteria, investigated as target sites for trimethoprim-like compounds, we also studied the reductases of some folate-requiring strains, since their folate auxotrophy suggested possible alterations in this enzyme as well. Dihydrofolate reductases from S. faecalis ATCC 8043 (syn. S. faecium var. durans), P. cerevisiae ATCC 8081, L. casei ATCC 7496, and L. arabinosus ATCC 8014, which, however, is not dependent on exogenous folate, were partially purified and their response to some structurally different inhibitors was compared to that of the enzymes from Escherichia coli B, Staphylococcus aureus, and also to rat liver enzyme as an example of an eucaryotic enzyme. Inhibition profiles, which have been successfully used for detecting subtle differences in dihydrofolate reductases of different origin (e.g., reference 6), revealed major differences in these enzymes. MATERIALS AND METHODSPropagation of celli. Cells were grown in batches of 200 ml up to 6 liters in either Erlenmeyer flasks under agitation or, in the case of the lactobacilli, without agitation. The following media were used: sensitivity test broth (Oxoid Ltd., London, England) for E. coli B, S. aureus, and S. faecalis; lactobacilli AOAC broth (Difco Laboratories, Detro...

Do Sulphonamide-trimethoprim Combinations Select Less Resistance to Trimethoprim than the Use of Trimethoprim Alone?

“…The molecular weights of these plasmid-determined enzymes are 32 000-37 000 and that of the normal chromosomal enzyme c. 18 000. Whilst there is some variation in the properties of TMP-sensitive DHFRs amongst different bacterial species (Then and Riggenbach, 1978), there are no TMP-sensitive DHFRs with molecular weights similar to those of the TMP-resistant DHFRs. It is not possible to envisage how the resistant enzyme could have evolved from the normal bacterial enzyme.…”

Low Trimethoprim Susceptibility of Anaerobic Bacteria Due to Insensitive Dihydrofolate Reductases

“…Double determinations were carried out as far as possible. A statistical comparison of the inhibition profiles obtained for these strains with six structurally different inhibitors was carried out essentially as described (18).…”

Trimethoprim and sulfonamide resistance

“…Endogenous resistance to TMP occurs among many different bacterial species. DHFRs from lactobacilli are thus less susceptible to TMP than the enzyme of E. coli, and the reductase from Pediococcus cerevisiae requires a 1,000-fold higher TMP concentration for 50% inhibition (173). Anaerobic bacteria like Bacteroides fragilis and Clostridium spp.…”