Photonic plasmid stability of transformed Salmonella typhimurium: a comparison of three unique plasmids.

BACKGROUND: Acquiring 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. RESULTS: In 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 x 10(7) to 1 x 10(9) 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 x 10(4) to 1 x 10(6) 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 x 10(3) to 1 x 10(5) CFU) low to high were different in the 96-well plate format (P < 0.05). CONCLUSION: These 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.

Hormonal profiles of late gestation ewes following intra-uterine inoculation with and without lux-modified Escherichia coli.

The objectives of these investigations were to develop an ovine model for Escherichia coli (E. coli)-induced preterm delivery, and monitor ewe hormonal response. EXP 1: Ewes (105 +/- 13 days of gestation) were allotted to the following intra-uterine inoculations: Saline-(CON; n=5); 1 x 10(6) CFU/ml (Low Treatment, LT; n=6); or 1 x 10(7) CFU/ml (High Treatment, HT; n=6) E. coli. Twenty-four h after inoculation, the HT ewes had increased (P<0.05) cortisol compared to LT and CON ewes, and HT and LT ewes had increased (P<0.05) progesterone compared to CON ewes. Preterm delivery was 33% for LT ewes and 0% for HT and CON ewes. EXP 2: Ewes (124 +/- 18 days of gestation) were allotted to the following intra-uterine inoculations using lux-modified E. coli: Trial-1: Luria Broth (LB; CT1; n=5); 4.0 x 10(6) CFU (n=5), 20.0 x 10(6) CFU (n=5); and Trial-2: LB (CT2; n=5), 1.2 x 10(6) CFU (n=5), and 5.6 x 10(6) CFU (n=5) E. coli-lux. Preterm delivery occurred between 48 and 120 h post-inoculation in 60, 25, 60 and 75% of ewes infected with 1.2, 4.0, 5.6, and 20 x 10(6) CFU, respectively. Serum cortisol and progesterone did not differ (P>0.05) between CT1 or CT2 and inoculated ewes. In summary, 25 to 75% of ewes inoculated preterm delivered. However, variable results in cortisol and progesterone profiles between Control and inoculated ewes were observed between the two studies.

Use of glycerol as an optical clearing agent for enhancing photonic transference and detection of Salmonella typhimurium through porcine skin.

The objective of this study was to evaluate glycerol (GLY) and GLY + dimethyl sulfoxide (DMSO) to increase photonic detection of transformed Salmonella typhimurium (S. typh-lux) through porcine skin. Skin was placed on 96-well plates containing S. typh-lux, imaged (5 min) using a CCD camera, and then completely immersed in PBS, GLY, DMSO, GLY+DMSO in a dose- and time-dependent manner and re-imaged (5 min). The percent of photonic emissions detected (treated or untreated skin relative to no skin controls) was used for analysis. Treatment for 4 h with 50% GLY-PBS and 50:30:20% GLY:DMSO:PBS increased photonic detection compared to untreated skin, 100% PBS, or 30:70% DMSO:PBS. Treatment with 50% GLY in the presence of 20 and 40% DMSO (v/v with PBS) increased photonic detection compared to 50% GLY alone and in the presence of 10% DMSO: 50% GLY (v/v with PBS). Data indicate that GLY and GLY+DMSO are effective optical clearing agents on porcine skin (2-3 mm thick) when treated for 4 h to increase detection of emitted photons. Clearing agents such as GLY have the potential to minimize effects of porcine skin tissue as one of the photon transmittance barriers (i.e., skin, fat, muscle, and visceral tissues) in vivo.

Development of a technique for serial bilateral renal biopsy in steers.

A biopsy procedure was developed to provide serial kidney samples from standing steers. Ten clinically normal steers were given intramuscular injections of gentamicin sulfate, 4 mg/kg body weight. Renal biopsy was performed at 5 separate times. After feed was withheld for 24 h, laparoscopic surgery was performed in standing stocks. Acepromazine, xylazine, and butorphanol were used for sedation and analgesia, and 2% lidocaine was used for local anesthesia. Two incisions approximately 2 cm long were made in the paralumbar fossa to allow for trocar introduction. The abdomen was insufflated with CO2 and, with endoscopic guidance, a biopsy forceps used to remove a kidney sample 2 to 3 mm in diameter, by either a left or a right abdominal approach. Each operation was recorded on videotape, and images were also captured with a digital medical device system. Respiration, heart rate, temperature, appetite, attitude, and postural positions were evaluated at 12, 24, 48, and 72 h after surgery. The 51 laparoscopic procedures provided 48 renal samples (approximately 100 mg each). The 1st and 2nd samples were from the right kidney, and the 3rd sample was from either the left or the right kidney; the 4th and 5th samples were from the left kidney. Adhesions made an approach from the right side difficult for the 3rd sample. No clinical changes were observed in 9 steers after the procedure. One steer died after the 3rd procedure owing to hemorrhage.

LC/MS/MS measurement of gentamicin in bovine plasma, urine, milk, and biopsy samples taken from kidneys of standing animals.

Methods for the measurement of gentamicin concentration in several bovine tissues were developed and validated. A novel liquid chromatographic (LC) technique employed trifluoroacetic acid in the mobile phase so that all gentamicin components co-eluted. Analytes were ionized by positive-ion pneumatically assisted electrospray and detected by selected reaction monitoring (SRM) with an LC-tandem mass spectrometer (LC/MS/MS). Calibration of plasma and urine samples was based on tobramycin internal standard. Calibration of milk and kidney samples was based on external standard, due to variability of tobramycin response in these matrices. The extraction technique employed treatment with aqueous trichloroacetic acid to both precipitate protein and liberate gentamicin from the matrix. Milk samples had to be defatted by centrifugation prior to extraction. Urine samples were further cleaned up with C-18 solid phase extraction (SPE). These methods were validated for use in several residue depletion studies (reported elsewhere) to monitor the depletion of gentamicin in tissues under various dosing conditions. The plasma method was calibrated from 1 to 5000 ng/mL in two ranges, with a limit of quantitation (LOQ) in the low range calculated at 3.3 ng/mL. The milk method was calibrated from 2.5 to 2500 ng/mL with an LOQ calculated at 4.5 ng/mL. The urine method was designed for use at low levels, and was calibrated from 1 to 100 ng/mL with an LOQ of 3.8 ng/mL. The kidney method was primarily designed for analysis of small samples (approximately 100mg). This method was calibrated from 10 to 50,000 ng/g with an LOQ of 26 ng/g.