Enrofloxacin metabolism by Mucor ramannianus was investigated as a model for the biotransformation of veterinary fluoroquinolones. Cultures grown in sucrose-peptone broth were dosed with enrofloxacin. After 21 days, 22% of the enrofloxacin remained. Three metabolites were identified: enrofloxacin N-oxide (62% of the total absorbance), N-acetylciprofloxacin (8.0%), and desethylene-enrofloxacin (3.5%).Fluoroquinolones are synthetic antimicrobial agents that are active against a broad spectrum of pathogenic gram-negative bacteria as well as some gram-positive bacteria and mycoplasmas (10). Several fluoroquinolones are used in clinical medicine, and enrofloxacin and sarafloxacin have been approved in the United States for veterinary use (4). The major metabolite of enrofloxacin in animals is ciprofloxacin, produced by N deethylation of the ethylpiperazine ring (21).The persistence of veterinary fluoroquinolones and the types of metabolites that result from their microbial conversion in the environment have been little known until recent years (6, 12, 22, 23; H. G. Wetzstein, Abstr. 99th Gen. Meet. Am. Soc. Microbiol. abstr. Q-262, p. 583, 1999). Currently, there is a question as to whether the use of fluoroquinolones in poultry causes the replacement of fluoroquinolone-sensitive coliform bacteria by resistant strains (2, 13).Cultures of wood-decaying basidiomycetes, including strains found in manure, have been shown to convert enrofloxacin to CO 2 and at least 11 other metabolites (12,22). Since other pathways are likely to be used by different organisms involved in the bioconversion of fluoroquinolones in the environment, we investigated the transformation of enrofloxacin by a typical zygomycetous soil fungus.Mucor ramannianus strain R-56, which had been isolated from a mushroom collected in a forest in Arkansas (17), was maintained on agar slants (14,18). Triplicate cultures for experiments were grown in sucrose-peptone broth (17) for 2 days. Enrofloxacin [1-cyclopropyl-7-(4-ethyl-1-piperazinyl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid], as the hydrochloride, was a gift from Bayer Corp., Shawnee, Kans. Enrofloxacin hydrochloride was dissolved in 20 mM aqueous KOH and filter sterilized; 1.0 ml was added to each flask to make a final concentration of 253 M enrofloxacin. The cultures were then incubated for an additional 21 days. Cultures without enrofloxacin and dosed, noninoculated controls were also incubated.Methylene chloride extracts of cultures and controls were analyzed by high-performance liquid chromatography (HPLC) (17), using a Phenomenex (Torrance, Calif.) Prodigy 5-m ODS-3 column (10 by 250 mm) with a water-acetic acid-methanol gradient (17). The flow rate was 2.5 ml min Ϫ1 ; metabolites were quantified by the peak areas at 280 nm.For direct exposure probe-electron ionization (DEP/EI) mass spectrometry (17), the quadrupole was scanned from m/z 50 to 650. For liquid chromatography-electrospray ionization (LC/ESI) mass spectrometry (17), a Vydac RP-18 pharmaceutical (5-m) microbore column (...

The presence of Burkholderia cepacia complex (BCC) strains has resulted in recalls of pharmaceutical products, since these opportunistic pathogens can cause serious infections. Rapid and sensitive diagnostic methods to detect BCC are crucial to determine contamination levels. We evaluated bacterial cultures, real-time PCR (qPCR), droplet digital PCR (ddPCR), and flow cytometry to detect BCC in nuclease-free water, in chlorhexidine gluconate (CHX) and benzalkonium chloride (BZK) solutions. Twenty BCC strains were each suspended (1, 10, 100, and 1000 CFU/ml) in autoclaved nuclease-free water, 10 μg/ml CHX, and 50 μg/ml BZK. Five replicates of each strain were tested at each concentration (20 strains × 4 concentrations × 5 replicates = 400 tests) to detect BCC using the aforementioned four methods. We demonstrated the potential of ddPCR and flow cytometry as more sensitive alternatives to culture-based methods to detect BCC in autoclaved nuclease-free water and antiseptics samples.

The metabolism of the fluoroquinolone drugs ciprofloxacin and norfloxacin by Pestalotiopsis guepini strain P-8 was investigated. Cultures were grown at 28 degrees C in sucrose/peptone broth for 18 days after dosing with ciprofloxacin (300 microM) or norfloxacin (313 microM). Four major metabolites were produced from each drug; and these were purified by high-performance liquid chromatography and identified by mass spectrometry and proton nuclear magnetic resonance spectroscopy. Ciprofloxacin metabolites included N-acetylciprofloxacin (52.0%), desethylene-N-acetylciprofloxacin (9.2%), N-formylciprofloxacin (4.2%), and 7-amino-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.3%). Norfloxacin metabolites included N-acetylnorfloxacin (55.4%), desethylene-N-acetylnorfloxacin (8.8%), N-formylnorfloxacin (3.6%), and 7-amino-1-ethyl-6-fluoro4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.1%). N-Formylciprofloxacin and the four transformation products from norfloxacin are all known to be mammalian metabolites.

Foodborne illnesses occur in both industrialized and developing countries, and may be increasing due to rapidly evolving food production practices. Yet some primary tools used to assess food safety are decades, if not centuries, old. To improve the time to result for food safety assessment a sensitive flow cytometer based system to detect microbial contamination was developed. By eliminating background fluorescence and improving signal to noise the assays accurately measure bacterial load or specifically identify pathogens. These assays provide results in minutes or, if sensitivity to one cell in a complex matrix is required, after several hours enrichment. Conventional assessments of food safety require 48 to 56 hours. The assays described within are linear over 5 orders of magnitude with results identical to culture plates, and report live and dead microorganisms. This system offers a powerful approach to real-time assessment of food safety, useful for industry self-monitoring and regulatory inspection.