Ciprofloxacin residues detection in cow milk: development of a novel and rapid optical b-Galactosidase-based screening assay

Screening methods (as defined in Commission Decision 2002/657 by European Union) play an important role as part of surveillance and monitoring at the primary production levels of the food supply chain (e.g. dairy supply chain). The optimizations of screening routinely used methods as well as the development of innovative methods are highly recommended by the European Community. Fluoroquinolones, specially ciprofloxacin, are among the most widely used antibacterial substances of synthesis in the veterinary practices for the treatment of bacterial infections in livestock. Various commercial kits are available for the detection of fluoroquinolones residues in foods, but these are time-consuming and mostly kits fails to detect residues at Maximum Residue Levels (MRLs) concentrations that are allowed by food safety authorities. We developed a novel and rapid optical microbiological screening assay for the detection of fluoroquinolone ciprofloxacin residues in cow milk samples. This screening assay is based on monitoring of Escherichia coli ATCC 11303 cell proliferation by optical measure of endogenous ?-galactosidase enzymatic activity. A linear correlation between the optical density of E. coli cultures (cell proliferation) and the induction of endogenous ?-galactosidase activity was experimentally established. ?-galactosidase enzymatic activity was determined using a chromogenic artificial substrate which changes the color of culture medium. The presence of ciprofloxacin residues in tested samples inhibits the E. coli cell proliferation which represents reduction in ?-galactosidase activity due to a lesser amount of enzyme (compared to control). The ?-galactosidase induction (normally obtained using isopropyl ?-D-1-thiogalactopyranoside (IPTG) was performed exploiting the lactose present in milk. The experimental results with lactose (from milk samples) were comparable to IPTG as synthetic inducer. The ciprofloxacin ?-galactosidase-based screening assay was performed by testing three different concentrations of ciprofloxacin in spiked cow milk samples and two different chromogenic ?-galactosidase artificial substrates, O-nitrophenyl-?-D-galactopyranoside (ONPG) and 5-bromo-4-cloro-3-indolil-beta-D-galattopiranoside (X-gal). The screening assay is able to detect ciprofloxacin residues within one hour at 1 MRL concentration using ONPG (cheaper than X-gal). This method can be adapted for the detection of ciprofloxacin residues in cow milk samples at primary milk production site.