Low Trimethoprim Susceptibility of Anaerobic Bacteria Due to Insensitive Dihydrofolate Reductases

Abstract

All the 28 Bacteroides fragilis strains investigated were susceptible to sulfamethoxazole (minimal inhibitory concentration < 16 Ag/ml) and resistant to trimethoprim (TMP; minimal inhibitory concentration > 4 ,ug/ml). Synergism between sulfamethoxazole and TMP was present in all strains at a ratio of 1:1. The few clostridia investigated proved more resistant to both compounds. Dihydrofolate reductases from B. fragilis, C. perfringens, and some other anaerobic species were isolated. Inhibition profiles with six structurally different inhibitors revealed major differences in all enzymes. For 50% inhibition, the enzyme from B. fragilis and all clostridia required concentrations of TMP which were between several hundredfold and 1,000-fold higher than those required for the enzyme of Escherichia coli, whereas the enzyme from Propionibacterium acnes only needed a threefold higher concentration. In vitro activities of TMP were seen to correspond to the activity at the enzymatic level in B. fragilis and P. acnes, but corresponded to a much lesser extent to the activity at the enzymatic level in clostridia, where a poor penetration is assumed to be involved. Dihydrofolate reductase inhibitors other than TMP were found to be as active as TMP both at the enzyme and in vitro. In B. fragilis, higher concentrations of exogenous thymidine were required for increasing the minimal inhibitory concentration of TMP than in E. coli and probably also in C. perfringens.Trimethoprim (TMP) is a highly active compound against a broad range of aerobic bacteria, since it interferes with the synthesis of tetrahydrofolate, derivatives of which are cofactors in several biochemical reactions, the most important being the synthesis of thymidylate (1,4). Among strictly anaerobic bacteria, however, at least partial resistance to TMP is a widespread feature, the basis of which is not yet understood. Clostridia are resistant in most cases (4,(6)(7)(8)19). Relatively high concentrations of TMP are needed (6,8,13,14,19) Media. The minimal inhibitory concentration (MIC) determinations were performed on DST agar supplemented with hemin chloride (5 lAg/ml) and menadione (0.5 Ag/ml). The concentration of thymidine in this medium is below the level needed to antagonize the action of antifolate compounds (16). In some cases, other media were used, as indicated below.MIC determinations. The agar disk method described by Linzenmeier and Neussel (12) for susceptibility testing of aerobic bacteria was modified for anaerobes. Stock solutions of TMP and sulfamethoxazole (SMZ) were appropriately diluted in distilled water. The compounds were incorporated into the medium by adding 13.5 ml of melted DST agar (supplemented) to 1.5 mnl of diluted solution of the compound in Folin tubes. The contents of the tubes were poured into 9-cm petri dishes and allowed to solidify.From the medium prepared in this way, agar disks with a surface of 0.8 cm2 were punched out and transferred to empty petri dishes, assembling a series of twofold dilution steps of the antibacterial c...

Susceptibility of Anaerobic Bacteria to Sulfamethoxazole/Trimethoprim and Routine Susceptibility Testing

“…The relative resistance of anaerobic bacteria to TMP is probably due to a lower susceptibility of the dihydrofolate reductases from anaerobes than those from facultatively anaerobic and aerobic bacteria. Inhibition of dihydrofolate reductases from B. fragilis and C. perfringens has been shown to require 100 to 1,000 times more TMP than the enzymes isolated from E. coli and Staphylococcus aureus (30).…”

“…Plates were also prepared in duplicate with the antimicrobial agents incorporated singly for determination of individual MICs and with SMZ and TMP (SMZ/TMP), in a ratio of 19:1, the ratio usually obtained in serum. Before use, the plates were dried at 37°C for 30 min.…”

Resistance among fecal flora of patients taking sulfamethoxazole-trimethoprim or trimethoprim alone

“…Within 10 min, the swab was squeezed out and removed, and serial dilutions were made for quantitative culture at 10-2, 10-3, lo-5, and 10-7. Serially diluted material was placed onto thymidine-free MacConkey agar prepared with Wellcotest base without antimicrobial agents, onto thymidine-free MacConkey agar with 4 ug of TMP per ml onto thymidine-free MacConkey agar with 21 Resistant aerobes were detected on the basis of growth on the antimicrobial agent-containing MacConkey plates. Quantitative fecal cultures were obtained in the same manner from controls, initially and after 6 weeks of no antimicrobial therapy.…”

Trimethoprim and sulfonamide resistance

“…are rather insusceptible to TMP. Purified preparations of DHFR from these strains required several hundredfold to 1,000-fold higher TMP concentrations than a similar preparation from E. coli for a 50% inhibition (170).…”