Postmortem photonic imaging of lux-modified Salmonella Typhimurium within the gastrointestinal tract of swine after oral inoculation in vivo1

Abstract

The study objective was to monitor Salmonella progression by photonic detection through segments of the gastrointestinal tract after oral inoculation. Pigs (~80 kg) were inoculated orally with 3.1 or 4.1 x 10(10) cfu of Salmonella Typhimurium transformed with plasmid pAK1-lux for a 6-h (n = 6) or 12-h (n = 6) incubation in vivo and then were killed for tissue harvest. Intestinal regions (duodenum, jejunum, ileum, large intestine) were divided into 5 replicates of 4 segments (5 cm) each for imaging. For each replicate, n = 2 segments of each region were intact, whereas n = 2 segments were opened to expose the digesta. Subsamples of digesta were analyzed to determine actual colony-forming units, and images were analyzed for relative light units per second. At 6 h, a greater (P < 0.05) concentration of emitting bacteria, and consequently a greater (P < 0.05) detection of photonic emissions, was observed in the small intestine than in the large intestine. The correlations (6 h) of photonic emissions in exposed segments to bacterial colony-forming units were r = 0.73, 0.62, 0.56, and 0.52 (P < 0.05) in duodenum, jejunum, ileum, and large intestine, respectively. Photonic emissions were greater (P < 0.05) in intact jejunum, ileum, and large intestine than in the duodenum after a 6-h incubation. At 12 h, a greater (P < 0.05) concentration of emitting bacteria in jejunum and ileum of exposed segments was observed than in duodenum and large intestine of exposed segments. Photonic emissions were greater in ileum than duodenum, jejunum, and large intestine of exposed segments (P < 0.05). The correlations (12 h) of photonic emissions in exposed segments to bacterial colony-forming units were r = 0.71 and 0.62 for jejunum and ileum, respectively (P < 0.05). At 12 h, a greater (P < 0.05) concentration of emitting bacteria in jejunum and ileum of intact segments was observed than in duodenum and large intestine. These data indicate that colony-forming units of introduced bacteria remained greater in the small intestine after 6- and 12-h incubations; we have determined that a minimum of 2.0 x 10(5) cfu generates detection through these tissues (~1.0 to 21.0 relative light units/s). This study demonstrates the feasibility of using biophotonics in research models ex vivo for monitoring the pathogenicity of Salmonella in swine, in place of, or in conjunction with, traditional microbiological assessments and whether a greater level of sensitivity of detection and correlation to actual bacterial concentrations can be achieved.

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 XEN plasmid has been introduced into E. coli strains previously and reported to emit photonic emissions within the mouse model (Harms et al, 2009). In terms of emission of photons over time, Moulton et al (2009b) reported a gradual 30% decline in bacterial emissions over an 8-d period (100% on d 0 to 66% on d 8) in the absence of antibiotic pressure. Moulton et al (2009b) suggested that the results of their study indicate that bioluminescent plasmids may permit real-time monitoring for 1-6 d in situations where antibiotic pressure may not be feasible.…”

“…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. 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.…”

“…For transformation of WT with the plasmid XEN (Caliper Life Sciences, Hopkinton, MA), cells were washed three times with 10% glycerol and electroporated with 50 ng of the XEN plasmid (Sambrook and Russell, 2001). The WT was transformed with PAK as previously described (Moulton et al, 2009b). Both transformed E. coli O157:H7 strains were selected for growth on TSA with 50 lg/mL of ampicillin (AMP) at 37°C; bioluminescence was confirmed by analyzing transformed strains with a Berthold/NightOwl camera equipped with the WinLight 32 software, version 2.51.11901 (Berthold Technologies, Oak Ridge, TN).…”

“…The ability to track bioluminescent pathogens in vivo through the gastrointestinal tract has previously been successful in pigs and mice (Burns-Guydish et al, 2005;Moulton et al, 2009b). Employing bioluminescence in live animal models will facilitate tracking of pathogens (Contag et al, 1997) and can indicate the physical location of these pathogens (Siragusa et al, 1999), thus enhancing the knowledge base of researchers with regard to pathogenic processes and transmission (Contag et al, 1995).…”