Bioluminescent Imaging of Bacterial Biofilm Infections In Vivo - Bacterial Pathogenesis: Methods and Protocols - page 213

progression and allows both response and/or relapse to antimicrobial agents
to be seen directly from intact live animals in real time, offering an excellent
preclinical strategy to assess disease response. Furthermore, this approach is
ideally suited to assess novel therapeutic agents or medical devices. This
will accelerate efforts to optimize lead compounds, especially at the animal
modeling step with respect to formulation, dose, or optimizing materials used
in medical implants.

2. Materials
1. Strains of bacteria: Staphylococcus aureus strain ATCC (American Type
Culture Collection, Manassas, VA) 12600 capsule type 3 strain, Pseudomonas
aeruginosa strain ATCC 19660, Proteus mirabilis strain ATCC 51286, and
Escherichia coli strain S17-1 pir pUT Amp R mini-Tn5 luxCDABE Tc R (M.K.
Winson, University of Nottingham, UK).
2. Plasmid pXen-5 (Xenogen Corp, Alameda, CA).
3. Ampicillin,

tetracycline, erythromycin, and kanamycin (Sigma-Aldrich, St.

Louis, MO).
4. MgSO 4 , glycerol, glucose, Luria-Betani (LB) broth, trypticase soy broth (TSB)
(Difco, Detroit, MI), and brain heart infusion (BHI) media (Becton Dickinson,
Franklin Lakes, NJ).
5. Ketamine and xylazine (Burns Vet Supply, Vancouver, WA).
6. GenePulser II and cuvettes (Bio-Rad, Richmond, CA).
7. Teflon intravenous catheter, 14-gauge (Abbocath-T; Burns Vet Supply).
8. Polyethylene PE 50 (OD 0.965 mm, ID 0.58 mm; Braintree Scientific, Inc,
Braintree, MA).
9. IVIS ® Imaging System and LivingImage ® software (Xenogen Corp).
10. Ultrasonic bath (VWR, San Francisco, CA).
11. Surgical staples, scissors, and blades (SPI Supplies, West Chester, PA).
12. Balb/C, CF-1, and CD-1 female mice weighing 18-30 g (Charles River,
Wilmington, MA).

3. Methods
3.1. Generation of Bioluminescent Bacteria
To maintain bioluminescence stably, all bacteria used for BPI studies were
transformed so that an optimized Photorhabdus luminescense lux operon is
integrated into their chromosome. These operons encode the genes necessary
for both luciferase and substrate production, enabling bioluminescence to be
produced by the transformants without the addition of exogenous products. Two
methodologies for stably transforming the particular Gram-negative and Gram-
positive bacteria used in the biofilm studies to a bioluminescent phenotype are
described below.