Optimization and validation of a simple method using P22::luxAB bacteriophage for rapid detection of Salmonella...

A simple method was developed for the fast and inexpensive detection of Salmonella Typhimurium using a recombinant P22::luxAB phage. All the steps from phage production to detection were considered. A strain of Salmonella Typhimurium harboring the prophage P22::luxAB was grown in batch culture to produce spontaneously the recombinant bacteriophage. Batch production to stationary phase was better for propagation of the phage and led to a total population of 4.3 x 10(9) (+/-4.3 x 10(9)) PFU/ml of P22, including only 1.4 x 10(6) (+/-1 x 10(6)) PFU/ml harboring the luxAB genes. After preenrichment, a simple four-step bioassay was tested and optimized for several parameters. The detection limit of the luminometer was only 5 x 10(2) (+/-1.75 x 10(2)) CFU Salmonella Typhimurium per ml, but increased to 1.5 x 10(4) (+/-1.17 x 10(4)) CFU Salmonella Typhimurium per ml when the cells were in a complex matrix. The detection limit after the preenrichment was 6.5 x 10(3) (+/-1.5 x 10(3)) CFU Salmonella Typhimurium per ml, but the detection limit after the preenrichment also increased markedly to 1.65 x 10(5) (+/-0.15 x 10(5)) CFU Salmonella Typhimurium per ml when Salmonella Typhimurium was in a complex matrix. Finally, the bioassay was applied to the detection of Salmonella Typhimurium LT2 in 14 different feed and environmental samples (including duck feed, litters, and feces) spiked either before or after the preenrichment process. It was possible to detect Salmonella Typhimurium LT2 in all samples within 16 h.