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Fungal transformation of an antimicrobial fluoroquinolone drug during growth on poultry litter materials

2004 Poultry Science Association, Inc.
Fungal Transformation of
an Antimicrobial Fluoroquinolone
Drug During Growth on Poultry
A. J. Williams,* I. A. Parshikov,† J. D. Moody,* T. M. Heinze,*
and J. B. Sutherland*,1
*National Center for Toxicological Research, U. S. FDA, Jefferson, Arkansas 72079; and †Department of Medicinal Chemistry, University of Mississippi, Oxford, Mississippi 38677 Primary Audience: Researchers, Veterinarians, Public Health Officials
The ability of a nonpathogenic fungus, Pestalotiopsis guepini, to metabolize fluoroquinolone
antimicrobial agents during growth on poultry litter materials was investigated. Sterilized rice
hulls, ground corncobs, and pine shavings in glass jars covered with foil were moistened with
sterile water and inoculated with P. guepini
. The litter materials then were dosed with norfloxacin
and incubated for 20 d. In rice-hull cultures, P. guepini
produced 4 metabolites: 7-amino-1-ethyl-
6-fluoro-4-oxo-1,4-dihydroquinolone-3-carboxylic acid, N
-formylnorfloxacin, N-acetylnorfloxacin,
and desethylene-N
-acetylnorfloxacin. In corncob cultures, the fungus produced N-formylnorfloxacin
and N
-acetylnorfloxacin. In pine-shavings cultures, there was little growth of the fungus and no
metabolism of norfloxacin. The results suggest that fungi that grow on poultry litter may degrade
residues of antimicrobial drugs.

Key words: fluoroquinolone, norfloxacin, poultry litter
2004 J. Appl. Poult. Res. 13:235–240
DESCRIPTION OF PROBLEM
that environment or are degraded by microor-ganisms that grow in the litter.
The use of antimicrobial agents in the poul- Norfloxacin, a fluoroquinolone antimicro- try industry to treat infections and promote bial agent, is used clinically for the treatment growth has frequently been associated with in- of urinary tract infections, bacterial enteritis,and eye infections [6, 7, 8]. The same drug is creases in bacterial resistance to clinically im- also used in poultry production in some coun- portant drugs [1, 2, 3, 4]. For instance, when tries for chronic respiratory diseases caused by fluoroquinolones are used, they may select for Mycoplasma synoviae and Escherichia coli [9, fluoroquinolone-resistant bacteria that can be 10, 11], although it is not registered for this found in poultry litter [5]. It is not known, purpose in the United States.
however, whether all of the antimicrobial drugs Poultry litter contains many bacteria, some that reach poultry litter persist indefinitely in of which may be resistant to multiple antibiot- 1 To whom correspondence should be addressed: [email protected]
JAPR: Research Report ics [5, 12, 13]. Although Enterococcus spp., Salmonella enterica, and other pathogenic bac- equipped with an HP 1090L/M liquid chroma- teria are sometimes found [13, 14, 15], they tograph. A 2.0 × 250 mm Prodigy column was usually are minor components of the total poul- used with a water and acetonitrile gradient con- try litter microbiota [16]. Saprobic fungi in the taining 0.1% formic acid [21]. Full scans were genera Aspergillus, Fusarium, Mucor, Penicil- acquired in the positive-ion electrospray mode, lium, and others are also found in poultry feed with the capillary exit voltage at either +100 and litter [17, 18, 19, 20]. The objective of this V or variable for molecular weight confirma- study was to determine whether poultry litter tion as the protonated molecule. The analysis materials could affect the degradation of nor- was repeated at +200 V to obtain fragments floxacin by Pestalotiopsis guepini, a fungus with [MH-H2O]+ as the base peak. Norfloxacin known to metabolize fluoroquinolones [21].
metabolites were identified by comparing re-tention times and mass spectra with those pub- MATERIALS AND METHODS
lished previously [21].
Typical poultry litter materials (10 g of rice Proton nuclear magnetic resonance (1H hulls, 10 g of pine shavings, or 20 g of ground NMR) spectral analyses were performed at 500 corncobs) were placed in 500-mL mason jars MHz on a Bruker AM500 NMR spectrometer and sterilized by autoclaving for 1 h on each [22] using deuterated methanol as the solvent.
of 2 successive days. Cultures of the fungus The results were compared with those pub- lished previously [21].
1325), grown on petri dishes of potato dextrose RESULTS AND DISCUSSION
agar, were macerated in sterile water using ablender. Each jar of litter material was inocu- Pestalotiopsis guepini grew well on rice lated with 10 mL of the blended mycelium.
hulls and corncobs but not on pine shavings.
Norfloxacin was dissolved in 2% aqueous KOH At 20 d, 4 metabolite peaks were detected by (100 mg of norfloxacin per mL) and filter-steri- HPLC in extracts from dosed rice-hull cultures lized, and then 1 mL of this solution was added that were not seen in extracts from control jars.
to each jar together with 40 mL of sterile water.
Two of the metabolite peaks were also detected Controls were prepared using each of the types in extracts from dosed corncob cultures. At 10 of litter material without the fungus or without and 14 d, smaller concentrations of the metabo- norfloxacin. The jars were incubated in the dark lites were observed. Some additional peaks at 28°C. On d 0, 10, 14, and 20, triplicate sets were seen in the corncob and pine-shavings of all cultures and controls were harvested, cultures and controls; the mass spectra of these filtered using glass wool, and extracted 3 times peaks (not shown) indicated that they were not with 100 mL of methylene chloride. The ex- tracts were combined and then evaporated in Mass and NMR spectroscopic analyses of vacuo. The residues were dissolved in 2.0 mL the peaks collected as they eluted from the of methanol for analysis.
HPLC column were used to identify 4 norflox- The extracts were analyzed for possible acin metabolites in the cultures of P. guepini norfloxacin metabolites by high-performance grown on rice hulls dosed with norfloxacin liquid chromatography (HPLC), using a Hew- (Figure 1A); the mass and NMR spectra were lett-Packard Series 1100 liquid chromatograph the same as those published previously [21].
with a Prodigy 5 µm ODS(3) 10.0 × 250 mm The structures of these compounds are shown column. The mobile phase consisted of a water in Figure 2. The major metabolite was 7- and methanol gradient containing 0.2% acetic acid [22] at a flow rate of 3.5 mL/min. The quinoline-3-carboxylic acid (I), and the other diode array detector was monitored at 280 nm, metabolites were N-formylnorfloxacin (III), N- and metabolite concentrations were estimated acetylnorfloxacin (IV), and desethylene-N-ace- from the total peak areas.
tylnorfloxacin (II). Two norfloxacin metabo- Liquid chromatography and mass spec- lites were identified in cultures of P. guepini trometry analyses were performed on a Hew- during growth on corncobs dosed with nor- WILLIAMS ET AL.: DRUG DEGRADATION IN LITTER FIGURE 1. HPLC chromatograms (280 nm) of extracts from cultures of Pestalotiopsis guepini grown for 20 d onpoultry litter materials dosed with norfloxacin, showing norfloxacin and fungal metabolites I to IV (A: rice hulls; B:corncobs; C: pine shavings). Peaks representing compounds that were shown by mass spectrometry to be unrelatedto norfloxacin are not numbered.
floxacin (Figure 1B); the major metabolite was The relative amounts of the norfloxacin me- N-formylnorfloxacin (III), and the minor one tabolites produced on poultry litter materials was N-acetylnorfloxacin (IV). Little growth of at 20 d were estimated from the total areas of the fungus was observed on pine shavings, and all the ultraviolet peaks in the HPLC chromato- no norfloxacin metabolites were detected (Fig- grams (Table 1). In rice-hull cultures, 18.4% of the total consisted of 7-amino-1-ethyl-6- JAPR: Research Report FIGURE 2. Structures of norfloxacin and the 4 metabolites produced from it by Pestalotiopsis guepini during growthon rice hulls dosed with norfloxacin [21].
is the predominant metabolite in broth cultures.
ylic acid (I), 0.5% was desethylene-N-acetyl- In rice-hull cultures, more of metabolite I (7- norfloxacin (II), 4.2% was N-formylnorfloxa- cin (III), and 2.3% was N-acetylnorfloxacin quinoline-3-carboxylic acid), which has lost (IV). In corncob cultures, 10.7% of the total the piperazine ring, and less of metabolites II was N-formylnorfloxacin (III), and 3.4% was and IV were formed. In corncob cultures, me- tabolites III (N-formylnorfloxacin) and IV (N- All 4 of the norfloxacin metabolites de- acetylnorfloxacin) were found, but there were tected in poultry litter materials are also pro- no products requiring cleavage of the pipera- duced in sucrose-peptone broth cultures with zine ring. Little growth of P. guepini and no norfloxacin [21], although N-acetylnorfloxacin metabolites were observed on pine shavings, WILLIAMS ET AL.: DRUG DEGRADATION IN LITTER TABLE 1. Amounts of each of the norfloxacin metabolites produced by Pestalotiopsis guepinii during growth for20 d on poultry litter materials dosed with norfloxacin Percentage of totalA represented by each metabolite carboxylic acid (I) AMeans and standard errors from triplicate cultures, based on the total integrated areas at 280 nm of all of the identifiedmetabolite peaks plus residual norfloxacin.
BND = not detected.
which contain stilbenes, resin acids, and other quinolone, an oxonorfloxacin, a methyl ester, compounds known to inhibit many fungi [23].
and an N-acetylethylenediamine-substituted Another fungus, Trichoderma viride, me- quinolone [26]. Since the known metabolites tabolizes norfloxacin to the conjugate 4- of fluoroquinolones are generally less active as antibacterial agents than the parent drugs [27], floxacin when grown in sucrose-peptone broth the fungal transformation of fluoroquinolones [24] and to the same conjugate plus N-acetyl- in poultry litter materials may reduce the selec- norfloxacin when grown on rice hulls [25]. The tive pressure on bacteria toward increased drug mammalian metabolites of norfloxacin include resistance. The type of litter materials used N-acetyl- and N-formylnorfloxacin, an ethyl- may affect the growth of fungi and the transfor- enediamine-substituted quinolone, an amino- mation of antimicrobial agents.
CONCLUSIONS AND APPLICATIONS
1. Two common poultry litter materials, rice hulls and corncobs, served as nutrients for the fungus Pestalotiopsis guepini and allowed it to transform added norfloxacin.
2. When grown on rice hulls dosed with norfloxacin, P. guepini produced 7-amino-1-ethyl-6- fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, N-formylnorfloxacin, N-acetylnor-floxacin, and desethylene-N-acetylnorfloxacin. When grown on corncobs, the fungus producedonly N-formylnorfloxacin and N-acetylnorfloxacin.
3. Pestalotiopsis guepini did not grow well on pine shavings, nor did it metabolize norfloxacin REFERENCES AND NOTES
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