Isolation of nitrofurantoin-resistant mutants of nitroreductase-producing Clostridium sp. strains from the human intestinal tract

Abstract

Five spontaneous nitrofurantoin-resistant mutants (one each of Clostridium leptum, Clostridium paraputrificum, two other Clostridium spp. strains from the human intestinal microflora, and Clostridium perfringens ATCC 3626) were selected by growth on a nitrofurantoin-containing medium. All of the Clostridium wild-type and mutant strains produced nitroreductase, as was shown by the conversion of 4-nitrobenzoic acid to 4-aminobenzoic acid. High-performance liquid chromatography (HPLC) analysis of the mutants during incubation with 50 microg of nitrofurantoin per ml showed the gradual disappearance of the nitrofurantoin peak. The nitrofurantoin peak also disappeared when cell-free supernatants instead of cultures of each of the resistant and wild-type bacteria were used, but it persisted if the cell-free supernatants had been inactivated by heat. At least two of the mutants converted nitrofurantoin to metabolites without antibacterial activity, as was shown by a bioassay with a nitrofurantoin-susceptible Bacillus sp. strain. Nitrofurantoin at a high concentration (50 microg/ml) continued to exert some toxicity, even on the resistant strains, as was evident from the longer lag phases. This study indicates that Clostridium strains can develop resistance to nitrofurantoin while retaining the ability to produce nitroreductase; the mutants metabolized nitrofurantoin to compounds without antibacterial activity.

FIG. 1

Effect of the concentration of nitrofurantoin on the growth of resistant strains of Clostridium species. (A) C. perfringens 3626NF; (B) C. paraputrificum NR1NF; (C) Clostridium sp. strain NR8NF. ○, no drug; •, 10 μg of nitrofurantoin per ml; □, 20 μg of nitrofurantoin per ml; ▪, 50 μg of nitrofurantoin per ml.

FIG. 2

HPLC elution profiles of cultures of a nitrofurantoin-resistant mutant, Clostridium sp. strain NR9NF, in BHI medium containing nitrofurantoin. (A) Culture inoculated with the mutant and sampled at zero time. (B) Culture inoculated with the mutant after incubation. (The elution profile of the noninoculated medium with nitrofurantoin after incubation for 24 h and that of nitrofurantoin after incubation with a heat-treated culture supernatant were both similar to that shown in panel A; the profile of the nitrofurantoin after incubation with an active culture supernatant was similar to that shown in panel B.)

FIG. 3

Areas of the nitrofurantoin peaks in HPLC profile after anaerobic incubation with overnight cultures of Clostridium sp. mutants or noninoculated BHI medium. ▪, C. paraputrificum NR1NF; , C. perfringens 3626NF; ▤, Clostridium sp. strain NR8NF; ▨, C. leptum NP15NF; □, Clostridium sp. strain NR9NF; ▩, BHI medium.

FIG. 4

(A) Number of surviving cells of a nitrofurantoin-sensitive bacterium (Bacillus sp. strain PS) before and after incubation with either BHI broth or cultures of nitrofurantoin-resistant strains of Clostridium species (grown with or without nitrofurantoin). ▪, BHI broth; □, BHI and nitrofurantoin; •, C. paraputrificum NR1; ○, C. paraputrificum NR1NF with nitrofurantoin; ✚, Clostridium sp. strain NR9; ×, Clostridium sp. strain NR9NF grown with nitrofurantoin. (B) Number of surviving cells of a nitrofurantoin-sensitive bacterium (Bacillus sp. strain PS) before and after incubation with either BHI broth or cultures of nitrofurantoin-resistant strains of Clostridium species (grown with or without nitrofurantoin). ▴, BHI broth; ▵, BHI and nitrofurantoin; ▪, C. perfringens ATCC 3626; □, C. perfringens 3626NF with nitrofurantoin; ✚, C. leptum NP15; ×, C. leptum NP15NF grown with nitrofurantoin; ○, Clostridium sp. strain NR8; •, Clostridium sp. strain NR8NF. The incubation of Bacillus sp. strain PS with the supernatants of nitrofurantoin-resistant bacteria in the absence of nitrofurantoin produced results identical to those obtained for parental strains (data not shown).