To construct a Klebsiella oxytoca (K. oxytoca)-pBBR1-lux strain that stably express luciferase (lux) gene, and to investigate the distribution of K. oxytoca in vivo and to evaluate the effect of different methods of sterilization.

The luciferase gene cluster (Lux A/B/C/D/E) of pBAV1k-T5-Lux plasmid was amplified, and the pBBR1 plasmid (pBBR1-lux) containing Lux A/B/C/D/E gene cluster was constructed; while Escherichia coli which expressed fluorescein gene group (E. coli-pBBR1-lux) was obtained. The expression of luciferase in E. coli-pBBR1-lux and K. oxytoca-pBBR1-lux were tested using luminescence microplate spectrophotometer. The pBBR1-lux plasmid was electroconverted to the receptive cells of K. oxytoca; after fluorescence intensity identification and three passages of the strain, K. oxytoca-pBBR1-lux which stably express lux genes were screened. Conjugated circular plasmid product was named pBBR1-lux. The value of Luminesence fluorescence signal of E. coli-pBBR1-lux increased significantly compared with E. coli control strain [15 345 (14 676, 18 654) vs. 63 (60, 82)], with significant difference (t = 21.14, P = 0.035). The value of Luminesence fluorescence signal of K. oxytoca-pBBR1-lux [399 303 (265 245, 617 192)] was significantly higher than that of the control strain K. oxytoca [83 (63.5, 86.75)], with significant difference (t = 7.07, P = 0.014). E. coli-pBBR1-lux and K. oxytoca-pBBR1-lux were both able to detect fluorescence signals on Veritas microplate photometer and small animal imager. According to 1︰10 dilution of 6 gradients, the fluorescence detection results showed that the Luminesence value decreased with the decreasing of colony concentration. After repeated passage pBBR1-lux luciferase could be stably expressed in K. oxytoca. Different physicochemical methods had different bactericidal effect on K. oxytoca-producing bacteria. Ultraviolet ray and 84 disinfectant (10%) were the most effective methods to inactivate K. oxytoca-producing bacteria.