Bioluminescent Imaging of Bacterial Biofilm Infections In Vivo - Bacterial Pathogenesis: Methods and Protocols - page 216

3.5. Soft-tissue Biofilm Infection
This device-related infection is established in the flanks of mice by subcu-
taneous implantation (17) of either pre- or in vivo -colonized catheter material
with biofilm forming Gram-positive or Gram-negative bioluminescent bacteria
(7) . The model is ideally suited for screening the development of biofilms
on medical device material and the effects of therapeutic agents to treat them
non-invasively without the need for exogenous sampling or culturing of the
pathogen ( see Fig. 1 ). Infections could be established with clinically relevant
doses as low as 10 3 CFUs pathogen per device with either Gram-positive or
Gram-negative pathogens. Moreover, subsequent disease progression could be
followed for weeks within the same animal.
This model is especially appealing for the analysis of in vivo efficacy of antibi-
otics against young as well as mature biofilms, post antibiotic effects, in vivo
resistance development, or in vivo fitness of resistant organisms (8,9,11,13) .
As the imaging procedure can be repeatedly performed over time without
killing the animal or removal of device for detachment of biofilm ( see Fig. 2
and Color Plate 7, following p. 46), this model, with several of the clinically
relevant features, has proven to have significant advantages over conventional
methods for studying foreign-body-related infections and treatment in vivo .

3.6. Experimental Model of Infection
1. Anesthetize Balb/C female mice weighing 18-22 g with Ketamine (100 mg/kg)
and Xylazine (5 mg/kg).
2. Shave their flanks and clean the skin with Betadyne and alcohol.
3. Make a 4-5 mm skin incision (as small as possible) and dissect to create a
subcutaneous tunnel.
4. Push a 1-cm segment of intravenous catheter with its associated bioluminescent
bacterial biofilm (e.g., Xen) through the incision into the tunnel (subcutaneous
tunnel usually created is ∼ 1.5 cm in length). One catheter segment is inserted on

each side of each animal.
5. Cover the incisions with intact skin, close with surgical staples, and disinfect the
skin.
6. Following surgery, place the mice on a warming pad until fully recovered.
7. Image animals using a highly sensitive low-light optical imaging system. (see
imaging procedure 3.12).
8. If post-implant infection is preferred, implant segments of sterile catheters as
above, then approximately 1 h after the implantation procedure, introduce a
defined quantities of bacterial suspension in 50 μL of PBS by injection into the
catheter lumen (10 3 -10 5 CFUs/catheter) via a 31-gauge needle.
Delaying the infection until after device implantation is useful to study factors
such as post-operative colonization of implanted device or the role of host