Degradation of the fluoroquinolone enrofloxacin by the brown rot fungus Gloeophyllum striatum: identification of metabolites

Abstract

The degradation of enrofloxacin, a fluoroquinolone antibacterial drug used in veterinary medicine, was investigated with the brown rot fungus Gloeophyllum striatum. After 8 weeks, mycelia suspended in a defined liquid medium had produced 27.3, 18.5, and 6.7% 14CO2 from [14C]enrofloxacin labeled either at position C-2, at position C-4, or in the piperazinyl moiety, respectively. Enrofloxacin, applied at 10 ppm, was transformed into metabolites already after about 1 week. The most stable intermediates present in 2-day-old supernatants were analyzed by high-performance liquid chromatography combined with electrospray ionization mass spectrometry. Eight of 11 proposed molecular structures could be confirmed by 1H nuclear magnetic resonance spectroscopy or by cochromatography with reference compounds. We identified (i) 3-, 6-, and 8-hydroxylated congeners of enrofloxacin, which have no or only very little residual antibacterial activity; (ii) 5,6- (or 6,8-), 5,8-, and 7,8-dihydroxylated congeners, which were prone to autoxidative transformation; (iii) an isatin-type compound as well as an anthranilic acid derivative, directly demonstrating cleavage of the heterocyclic core of enrofloxacin; and (iv) 1-ethylpiperazine, the 7-amino congener, and desethylene-enrofloxacin, representing both elimination and degradation of the piperazinyl moiety. The pattern of metabolites implies four principle routes of degradation which might be simultaneously employed. Each route, initiated by either oxidative decarboxylation, defluorination, hydroxylation at C-8, or oxidation of the piperazinyl moiety, may reflect an initial attack by hydroxyl radicals at a different site of the drug. During chemical degradation of [4-14C]enrofloxacin with Fenton's reagent, five confirmatory metabolites, contained in groups i and iv, were identified. These findings provide new evidence in support of the hypothesis that brown rot fungi may be capable of producing hydroxyl radicals, which could be utilized to degrade wood and certain xenobiotics.