|Lay Jr, Donald|
Submitted to: American Society of Animal Science
Publication Type: Abstract Only
Publication Acceptance Date: 12/15/2005
Publication Date: 2/4/2006
Citation: Williams, E., Moulton, K., Moore, D., Mcgee, M., Lovell, F., Couvillion, S., Ryan, P., Lay Jr, D.C., Willard, S. 2006. Photonic properties of transformed salmonella typhimurium: plasmid stability and concentration dependency. American Society of Animal Science.
Technical Abstract: Understanding the photonic properties of transformed bacteria for use in biophotonic studies of bacterial tracking in vivo is critical to experimental paradigm development. The objectives of this study were to determine: (1) the stability and photonic characteristics of transformed Salmonella typhimurium (S. typh-lux) using two different plasmids; and (2) the relationship between photonic emissions and concentrations of S. typh-lux. S. typh-lux isolates (porcine) were electroporated with either pCGLS-1 or pAK1-lux plasmids. S. typh-lux was then grown over a 24 h period in LB broth or LB broth (10 ml) plus ampicillin (AMP; 50 mg/ml) and placed in an incubated shaker. After 24 h, inoculums were placed into a 96-well plate for imaging (100 ul; n = 8 replicates per plate among 4 replicate experiments). Photonic imaging was conducted for 5 min and emissions quantified (photons/pixel/sec; PPS). Inoculums were plated on Brilliant Green agar plates (with and without AMP), colony numbers counted (CFU) and plates imaged for the ratio of non-emitting to emitting colonies. Inoculums were sub-cultured daily from d 0 to 14 with and without AMP selection to determine photonic stability between the two plasmids over time. Serial dilutions with the two plasmids up to 15.7 x 109 CFU/ml were evaluated to examine the relationship between CFU and photonic emissions. In the presence of AMP, S. typh-lux with the pCGLS-1 and pAK1-lux plasmids remained 100% emitting over the entire 15-d study. Photon emitters of S. typh-lux with pCGLS-1 and pAK1-lux without AMP selection both decreased over time (P < 0.01), representing only 25.4 ± 11.6% and 24.9 ± 8.5%, respectively, of the bacterial population by d 14 (plasmid: P > 0.10). S. typh-lux transformed with the pCGLS-1 plasmid exhibited similar (P > 0.10) photonic emissions as the pAK1-lux plasmid at similar CFU concentrations up to 100 x 106 CFU/ml (an apparent threshold for the imaging system based on PPS). These data characterize the photon emitting and stability properties for S. typh-lux transformed with two different photon generating plasmids for use with in vivo biophotonic paradigms.