Transformation of the antibacterial agent norfloxacin by environmental mycobacteria

Abstract

Because fluoroquinolone antimicrobial agents may be released into the environment, the potential for environmental bacteria to biotransform these drugs was investigated. Eight Mycobacterium sp. cultures in a sorbitol-yeast extract medium were dosed with 100 microg ml(-1) of norfloxacin and incubated for 7 days. The MICs of norfloxacin for these strains, tested by an agar dilution method, were 1.6 to 25 microg ml(-1). Cultures were extracted with ethyl acetate, and potential metabolites in the extracts were purified by high-performance liquid chromatography. The metabolites were identified using mass spectrometry and nuclear magnetic resonance spectroscopy. N-Acetylnorfloxacin (5 to 50% of the total absorbance at 280 nm) was produced by the eight Mycobacterium strains. N-Nitrosonorfloxacin (5 to 30% of the total absorbance) was also produced by Mycobacterium sp. strain PYR100 and Mycobacterium gilvum PYR-GCK. The MICs of N-nitrosonorfloxacin and N-acetylnorfloxacin were 2- to 38- and 4- to 1,000-fold higher, respectively, than those of norfloxacin for several different bacteria, including the two strains that produced both metabolites. Although N-nitrosonorfloxacin had less antibacterial activity, nitrosamines are potentially carcinogenic. The biotransformation of fluoroquinolones by mycobacteria may serve as a resistance mechanism.

FIG. 1.

HPLC elution profiles of ethyl acetate extracts from Mycobacterium sp. strain 7E1B1W (A), M. gilvum PYR-GCK (B), and Mycobacterium sp. strain PYR100 (C), all dosed with norfloxacin (NFX). M1 and M2, metabolites 1 and 2.

FIG. 2.

Product-ion mass spectra of norfloxacin metabolites with argon collision gas at 1.5 mtorr. (A) Metabolite 1 from Mycobacterium sp. strain 7E1B1W. The parent ion m/z is 362, and the collision energy is 35 eV. (B) Metabolite 2 from M. gilvum PYR-GCK. The parent ion m/z is 349, and the collision energy is 25 eV.

FIG. 3.

Norfloxacin (with positions numbered) and the metabolites produced by Mycobacterium spp. R stands for H in norfloxacin, COCH₃ in N-acetylnorfloxacin, and NO in N-nitrosonorfloxacin.