Detection of tetracyclines with luminescent bacterial strains.

The performance of two bioluminescent Escherichia coli K-12 strains for the specific detection of the tetracycline family of antimicrobial agents was compared, and the analytical applicability of one of the strains was preliminarily evaluated. One sensor plasmid contained the bacterial luciferase operon of Photorhabdus luminescens under the control of the tetracycline-responsive element from transposon Tn10 (15). An analogous plasmid construction with firefly (Photinus pyralis) luciferase reporter gene was constructed, and these two divergent tetracycline-inducible light-emitting systems were compared for their suitability for the qualitative detection of tetracyclines. Both sensor strains behaved in a similar manner kinetically, and the most sensitive tetracycline response for both sensor strains was achieved in 90-120 min by performing the assay at 37 degrees C. The sensor strain containing the bacterial luciferase operon responded slightly more sensitively to different tetracyclines than the strain containing firefly luciferase gene. The sensor bacteria retained their inducibility in lyophilization, and freeze-dried cells detected tetracyclines as sensitively as freshly cultivated sensor cells. The preliminary results from the analysis of tetracycline-spiked pork serum samples indicated that these sensor bacteria could be used to screen veterinary samples for tetracycline residues in real-time.

A group-specific microbiological test for the detection of tetracycline residues in raw milk.

The potentiality of using a luminescent Escherichia coli strain for the specific detection of tetracycline residues in raw bovine milk was investigated. The sensor cells contain a reporter plasmid carrying the bacterial luciferase operon of Photorhabdus luminescens under the control of the tetracycline responsive control region from transposon Tn10. Incubation of the cells with the sample containing tetracyclines increases the light emission of the sensor cells. The most sensitive tetracycline detection was achieved in 120 min and by using CDTA as a chelating agent in the assay. Heat-treatment of milk before the assay decreased the variations in background luminescence signals and in tetracycline-induced luminescence between different milk samples. The detection limits for tetracycline, oxytetracycline, chlortetracycline, doxycycline, methacycline, demeclocycline, and minocycline were between 2 and 35 ng/mL. Nontetracycline antibiotics did not significantly interfere with the detection of tetracyclines.

Qualitative detection of tetracycline residues in milk with a luminescence-based microbial method: the effect of milk composition and assay performance in relation to an immunoassay and a microbial inhibition assay.

Performance of Tet-Lux, a newly developed microbiological test for the detection of tetracycline residues in raw milk, based on tetracycline-controlled luminescence activation of the test bacteria, was evaluated in bovine milks with variable amounts of somatic cells, bacteria, fat, protein, and natural inhibitory compounds. The sensitivity of Tet-Lux was also compared to a commercially available tetracycline immunoassay (Snap, Idexx Laboratories Inc.) and to a microbial inhibition test (Delvotest SP, Gist-Brogades). There were slight differences in the luminescence signals between different milk samples, but no single factor could be pointed out to be responsible for them. There appeared to be a modest inverse relationship between luminescence and increasing fat and protein content. The amount of somatic cells, bacteria, and the natural inhibitors lysozyme and lactoferrin did not affect the luminescence response. The test fulfilled the sensitivity requirement specified by the European Union (maximum residue limit 100 ng/ml for tetracyclines). The Tet-Lux test was clearly more sensitive to all tetracyclines tested (oxytetracycline, tetracycline, chlortetracycline, doxycycline, demeclocycline, methacycline, minocycline) than Delvotest SP, and for five tetracyclines out of seven more sensitive than Snap. The test provides a fast, simple, and robust microbial method for the qualitative detection of tetracycline residues in milk.

A recombinant Escherichia coli sensor strain for the detection of tetracyclines.

A bioluminescent Escherichia coli K-12 strain for the specific detection of the tetracycline group of antibiotics is described. A sensor plasmid, containing five genes from bacterial luciferase operon of Photorhabdus luminescens inserted under the control of tetracycline-responsive elements of the transposon Tn10, was constructed. Usage of the full-length luciferase operon in the sensor resulted in tetracycline-dependent light production without additions, i.e., self-luminescent phenotype, since all the substrates were intrinsically produced by the recombinant organism. The time needed for optimal induction of light emission was 90 min. Maximal induction of approximately 100-fold over uninduced levels by using 20 ng of tetracycline, and picomole sensitivities for the seven different tetracyclines tested, were obtained without added Mg2+ ions. The higher the pH and the magnesium ion concentration in the assay medium the higher was the amount of membrane-impermeable tetracycline-Mg2+ chelate complex. In consequence, by adjusting the pH and the Mg2+ ion concentration, the sensitivity of the assay can be modified for different analytical purposes. Different non-tetracycline antibiotics did not cause induction of light emission.