The basic metabolic pathway of PRA, a new veterinary FQ antibacterial drug, in the brown rot fungus G. striatum
was similar to schemes established for other FQs such as enrofloxacin, ciprofloxacin and moxifloxacin. Hydroxylated primary metabolites of PRA, each representing a different class of compounds (Figure ), were generated by hydroxyl radical-based decarboxylation (F-1), defluorination (F-2) and elimination of CN (F-6). The definitive identification of a catechol-type FQ congener, compound F-5, carrying one hydroxyl group each at C-5 and C-6, is described here for the first time. This was facilitated by the CN substituent blocking C-8, in contrast to F-5 of enrofloxacin Wetzstein et al. 1997
or ciprofloxacin Wetzstein et al. 1999
, for which hydroxylation of position C-5 was indistinguishable from hydroxylation of C-8. Degradation of the amine substituent is represented by F-9.
The identification of metabolite F-13, consisting of the cyclopropyl-substituted pyridone part and C-atoms 7 and 8 of PRA, now linked by a double bond and carrying a hydroxyl and the CN group, respectively, proved fungal cleavage of the aromatic FQ core for the first time. The presence of a conjugated CN group was verified by IR-spectroscopy. Being part of a mesomeric system, the CN group is likely to have stabilized metabolite F-13 sufficiently as to permit its isolation and structure elucidation. The most likely intermediates, connecting F-5 with F-13 (Figure ), imply: (i) hydroxyl radical-based elimination of the intact amine moiety (F-10), a reaction already observed for enrofloxacin and ciprofloxacin Wetzstein et al. 1997
; (ii) twofold oxidation of the resulting pyrogallol-type intermediate and the formation of a cis,cis
-muconic acid-type analog; and (iii) spontaneous twofold decarboxylation, finally providing F-13. Metabolite F-13 adds a unique type of compound to the plethora of 137 known metabolites of enrofloxacin (including the ethylpiperazine residue and CO2
) produced by basidiomycetous fungi Karl et al. 2006
;Wetzstein et al. 2006
Six major metabolites of enrofloxacin and, in particular, 8-OH-PRA (F-6) have been shown to essentially have lost antibacterial activity Wetzstein et al. 2009
;Wetzstein and Hallenbach 2011
. Most recent observations suggest that minimum inhibitory concentrations of 8-OH-PRA may have been slightly (less than twofold) overestimated, due to its instability at 37°C, resulting in a half-life of about 2 days (Wetzstein H-G, unpublished data). Regardless, FQ residues such as those described herein appear to be unlikely to pose a significant risk due to selection of resistance in agricultural soils Wetzstein et al. 2009
;Baquero et al. 2011
Recently, metabolites of norfloxacin hydroxylated at position C-6 and C-8 have been reported to be formed by Microbacterium
spp. isolated from wastewater Kim et al. 2011
. Moreover, from 8-OH-norfloxacin, C-8 may be eliminated by Candida palmioleophila
LA-1 Kim et al., Abstr 111th Annu Gen Meet Am Soc Microbiol, abstr. Q-2943, 2011
. The corresponding metabolite should be indicative of a mechanism of aromatic ring cleavage different from that believed to be observed in this study.
Metabolic inactivation of FQs in mammals predominantly comprises glucuronidation of the carboxyl group and sulfation of an appropriate secondary amine function, if present in the amine substituent. Furthermore, N
-4'-dealkylation, formylation and oxide formation as well as partial degradation of the C-7 amine substituent have been observed. However, core-hydroxylated metabolites were not reported, as reviewed by Dalhoff and Bergan (1998)
. Another major mechanism of FQ inactivation is N-
4'-acetylation, in case of enrofloxacin following N
-4'-deethylation, as catalyzed by the Zygomycete Mucor ramannianus Parshikov et al. 2000
. For G. striatum
, only O
-acetylated congeners have been observed, exemplified by metabolites 13 and 62 described by Karl et al. (2006)
. Chemically synthesized N
-acetyl-PRA provides for MICs similar to those of 8-OH-PRA (Wetzstein H-G, unpublished observation). However, it is unknown, whether PRA could serve as a substrate for the bacterial enzymes yet described.
Most notable, acetylation of the piperazine residue of ciprofloxacin or norfloxacin by environmental strains of Mycobacterium Adjei et al. 2006a
has not yet been found in clinical strains. Furthermore, a FQ-resistant strain of E. coli
, isolated from sewage sludge, contained the aminoglycoside transacetylase gene aac(6')-Ib-cr
and was capable of modifying ciprofloxacin by acetylation Jung et al. 2009
. This activity was first observed to be a plasmid-encoded FQ-resistance factor in Gram-negative species Robicsek et al. 2006
-acetylation as well as N-oxide formation Parshikov et al. 2000
;Karl et al. 2006
eliminates the positive charge of the amine residue, present at physiological pH. The resulting FQ congener is negatively charged, thus drug accumulation into the cytoplasm may be restricted or even prevented.
Strangely enough, the apparently complex degradation scheme described for enrofloxacin Karl et al. 2006
;Wetzstein et al. 2006
has to be considered a relatively simple example, compared to degradation patterns to be expected for PRA, moxifloxacin and any other FQ with a more complex amine substituent. Giving rise to the variety of constitutional isomers, the number of discernible H-atoms in the amine moiety, potentially to be replaced by a hydroxyl group, amounts to four for enrofloxacin, but already to twelve for the pyrrolidinopiperidine residue of PRA; this structural feature extends to metabolites carrying a combination of a hydroxyl and a keto group or even a cleaved amine moiety. Hence, definitive structure elucidation of such metabolites would have to be based on isolated compounds and required a formidable analytical effort.
In independent studies assessing the chemical degradation of enrofloxacin, ciprofloxacin or other FQs, cleavage of the aromatic core could not yet be proved either, if based on metabolite identification. Work on ciprofloxacin confirmed several key metabolites observed with G. striatum
: (i) F-1, F-2, F-6 and F-9 in a membrane anodic Fenton-type system (Xiao et al. 
; see also references 19, 22 and 25, therein); (ii) isatin and anthranilic acid-type metabolites formed during ozonation (Dewitte et al. 2008
; and (iii) metabolites indicating degradation of the amine substituent by hydroxyl radicals, generated upon UV-irradiation of TiO2 Paul et al. 2007
. Under the latter conditions, cleavage of the cyclopropyl moiety was observed as well. This leaves unexplored fungal degradation of the cyclopropyl-substituent, which, however, is a natural product found, e.g., in the lipids of E. coli Goldfine 1982
. Two decades after the emergence of the notion of the non-biodegradability of FQs, briefly reviewed by Wetzstein et al. (2009)
, fungal degradation of all key structural elements of a FQ, in particular of the aromatic core, now has been proven.