Photonic plasmid stability of transformed Salmonella Typhimurium: A comparison of three unique plasmids

Abstract

BackgroundAcquiring a highly stable photonic plasmid in transformed Salmonella Typhimurium for use in biophotonic studies of bacterial tracking in vivo is critical to experimental paradigm development. The objective of this study was to determine stability of transformed Salmonella Typhimurium (S. typh-lux) using three different plasmids and characterize their respective photonic properties.ResultsIn presence of ampicillin (AMP), S. typh-lux with pCGLS-1, pAK1-lux and pXEN-1 plasmids exhibited 100% photon-emitting colonies over a 10-d study period. Photon emitters of S. typh-lux with pCGLS-1, pAK1-lux and pXEN-1 without AMP selection decreased over time (P < 0.05), representing only 11 ± 1%, 35 ± 1% and 43 ± 1%, respectively, of original photon emitting properties of the bacterial population by d 10. Photonic emissions were positively correlated with bacterial concentration (P < 0.05) for pAK1-lux, pCGLS-1 and pXEN-1 (r = 0.96, 0.98 and 0.82, respectively). When stratified by high, medium and low density bacteria concentrations, photonic emissions for high density populations containing pAK1-lux, pCGLS-1 and pXEN-1 resulted in differences of photonic emissions across a range of bacterial concentrations (1 × 107 to 1 × 109 CFU, P < 0.05) with positive correlations (P < 0.05) of (r = 0.72, 0.46 and 0.72, respectively). The correlation of photonic emissions with bacterial concentrations for samples with medium and low density bacteria (pAK1-lux, pCGLS-1, and pXEN-1 plasmids) imaged in tubes were also positively correlated (medium; r = 0.69, 0.49, 0.46, low; r = 0.90, 0.71, 0.68, respectively; P > 0.05); although photonic emissions across a range of bacterial concentrations were not different (1 × 104 to 1 × 106 CFU, P > 0.05). For very low density bacterial concentrations imaged in 96 well plates photonic emissions were positively correlated with bacterial concentration (P < 0.05) for pAK1-lux, pCGLS-1, and pXEN-1 plasmids (r = 0.99, 0.99, and 0.96, respectively), and photonic emissions across a range of bacterial concentrations (1 × 103 to 1 × 105 CFU) low to high were different in the 96-well plate format (P < 0.05).ConclusionThese data characterize photon stability properties for S. typh-lux transformed with three different photon generating plasmids that may facilitate real-time Salmonella tracking using in vivo or in situ biophotonic paradigms.

Survival ofEscherichia coliO157:H7 Transformed with Either the pAK1-luxor pXEN-13 Plasmids inIn VitroBovine Ruminal and Fecal Microbial Fermentations

“…For a period of 4 d, there was no difference in the decline with or without antibiotic pressure; however, after 6 d, there was a greater decline in emissions for bacteria without antibiotic pressure. In a similar study, Moulton et al (2009b), evaluating various plasmids (pCGLS-1, pAK1-lux, and pXEN-1) within Salmonella Typhimurium, reported a continual decline in percent of emissions for all three plasmids (very similar to Moulton et al, 2009a).…”

“…The PAK plasmid was developed as a wide range host plasmid for gram-negative bacteria (Karsi et al, 2006). Research with this plasmid has suggested stability and luminescence without antibiotic pressure for at least 8 d, though the percentage of photonic emissions decreases past 0 d (Moulton et al, 2009a). The XEN plasmid carries the original Photorhabdus luminescens operon for production of luminescence in gram-negative bacteria (Harms et al, 2009).…”

“…Additionally, the stability of these plasmids within E. coli MM294 and Salmonella Typhimurium has also been evaluated. A study conducted by Moulton et al (2009a) with E. coli MM294 reported a continual decline in the percentage of bacteria emissions from 100% to 84% over a 24-h period without antibiotic pressure and a decline from 100% to 66% over an 8-day (d) period (i.e., a 30% loss in emitting bacteria in the absence of antibiotic selection). Moulton et al (2009b) also reported that with or without antibiotic pressure, there was a continual decline in emissions.…”

HORSE SPECIES SYMPOSIUM: A novel approach to monitoring pathogen progression during uterine and placental infection in the mare using bioluminescence imaging technology and lux-modified bacteria1,2

“…The use of bioluminescent bacteria, such as E. coli-pAKl-lux or E. coZi!-Xenl4, coupled with bioluminescence imaging technologies, may represent an efficient model for achieving a greater understanding of the pathogenesis of uterine and placental infections in domestic species. Some studies have established positive relationships between bioluminescent signals and bacterial populations for gram-negative bacteria (r = 0.99; Kadurugamuwa et al, 2005), whereas other investigators have described and validated bioluminescent bacterial models, both in vitro (Moulton et al, 2006(Moulton et al, , 2009c and ex vivo (Moulton et al, 2009a,d;Curbelo et al, 2010). More recently, studies have reported the use of bioluminescent reporters and biophotonic imaging technology to study mechanisms of pathogenesis of such pathogens as Staphylococcus aureus (Kuklin et al, 2003), Aspergillus fmnigatus (Brock et al, 2008), Listeria monocytogenes (Konjufca and Miller, 2009), E. coli (Foucault et al, 2010), and Mycobacterium tuberculosis (Andreu et al, 2010); however, these studies have focused on the use of the mouse model.…”

“…For example, E. coli MM294 (no. 33625; American Type Culture Collection, Manassas, VA) has been modified with pAKl-lux plasmid, which contains an antibiotic-resistant gene cassette combined with the lux gene (Moulton et al, 2008(Moulton et al, , 2009c. The plasmid (11,904 bp) used in this instance is a broad-host-raiige cloning vector with numerous plasmid replicons containing the lux operon (Frackman et al, 1990).…”