Modification of norfloxacin by a Microbacterium sp. strain isolated from a wastewater treatment plant

Abstract

Antimicrobial residues found in municipal wastewater may increase selective pressure on microorganisms for development of resistance, but studies with mixed microbial cultures derived from wastewater have suggested that some bacteria are able to inactivate fluoroquinolones. Medium containing N-phenylpiperazine and inoculated with wastewater was used to enrich fluoroquinolone-modifying bacteria. One bacterial strain isolated from an enrichment culture was identified by 16S rRNA gene sequence analysis as a Microbacterium sp. similar to a plant growth-promoting bacterium, Microbacterium azadirachtae (99.70%), and a nematode pathogen, "M. nematophilum" (99.02%). During growth in medium with norfloxacin, this strain produced four metabolites, which were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) analyses as 8-hydroxynorfloxacin, 6-defluoro-6-hydroxynorfloxacin, desethylene norfloxacin, and N-acetylnorfloxacin. The production of the first three metabolites was enhanced by ascorbic acid and nitrate, but it was inhibited by phosphate, amino acids, mannitol, formate, and thiourea. In contrast, N-acetylnorfloxacin was most abundant in cultures supplemented with amino acids. This is the first report of defluorination and hydroxylation of a fluoroquinolone by an isolated bacterial strain. The results suggest that some bacteria may degrade fluoroquinolones in wastewater to metabolites with less antibacterial activity that could be subject to further degradation by other microorganisms.

Fig. 1.

Phylogenetic tree of Microbacterium sp. strain 4N2-2 based on 16S rRNA gene sequences constructed by the neighbor-joining method. Microbacterium barkeri DSM20145^T (GenBank accession no. X77446) was used as the outgroup (not shown). Percentages at the nodes are the levels of bootstrap support (>50%) from 1,000 resampled data sets. The solid circles indicate that the corresponding nodes (groupings) were also recovered in the maximum parsimony tree, and the bar represents 0.005 nucleotide substitution per position.

Fig. 2.

HPLC elution profiles showing norfloxacin (Nfx) and metabolites produced in 14-day cultures of Microbacterium sp. strain 4N2-2. Cultures were grown in either OM medium with ascorbic acid (A and B) or 2.0 CM medium (C).

Fig. 3.

Degradation of norfloxacin and production of metabolites by Microbacterium sp. strain 4N2-2 after addition of ascorbic acid to OM medium. (A) Disappearance of norfloxacin (▪; 280 nm) and production of 8-hydroxynorfloxacin (white bars; 240 nm), 6-defluoro-6-hydroxynorfloxacin (gray bars; 280 nm), and desethylene norfloxacin (black bars; 280 nm) were measured in 14-day cultures, with ascorbic acid added at 6 days. (B) Disappearance of norfloxacin (▪; 280 nm) and production of 8-hydroxynorfloxacin (□; 240 nm), 6-defluoro-6-hydroxynorfloxacin (▴; 280 nm), and desethylene norfloxacin (▵; 280 nm) were monitored over time in the presence of 0.5 mM ascorbic acid (arrow) added at 6 days.

Fig. 4.

Inhibition of production of hydroxylated norfloxacin metabolites by free radical scavengers. Production of 8-hydroxynorfloxacin (white bars; 240 nm), 6-defluoro-6-hydroxynorfloxacin (gray bars; 280 nm), and desethylene norfloxacin (black bars; 280 nm) was measured in 14-day cultures with various free radical scavengers. Sodium formate, thiourea, and mannitol (50 mM) were introduced to cultures in OM medium with ascorbic acid or NaNO₃.

Fig. 5.

Production of metabolites of norfloxacin by Microbacterium sp. strain 4N2-2. (A) Decreases of norfloxacin (▪; 280 nm) and production of N-acetylnorfloxacin (⧫; 280 nm) in 2.0 CM medium were monitored by HPLC over time. The arrow at 7 days indicates the addition of ascorbic acid. (B) The amounts of 8-hydroxynorfloxacin (white bars; 240 nm), 6-defluoro-6-hydroxynorfloxacin (gray bars; 280 nm), desethylene norfloxacin (black bars; 280 nm), and N-acetylnorfloxacin (striped bars; 280 nm) were measured in 14-day cultures of OM medium and minimal medium supplemented with 0.05, 0.5, and 2.0 g liter⁻¹ Casamino Acids (0.05 CM medium, 0.5 CM medium, and 2.0 CM medium, respectively).

Fig. 6.

Proposed scheme for norfloxacin metabolism in cultures of Microbacterium sp. strain 4N2-2.