Pilot Study

In the core project of this research, the goal is to identify the antimicrobial ability of a coated device to clear or prevent a urinary tract infection from an inoculated individual in an in vivo animal model. The control group to which we will be comparing our results should therefore ideally have a urinary tract infection during the experiment, which will be induced at time of device placement. The pilot study was performed to identify the ideal E. coli load inoculum that would induce an infection in the control group without inducing urosepsis. If an animal with an uncoated device in situ can clear the infection, we hypothesized that it should be able to clear that infection at least equally as easy or easier with our experimentally coated stent in situ. We used Escherichia coli GR12, a bacterial strain initially isolated from a patient with pyelonephritis (Hagberg et al., 1983; Svanborg Edén et al., 1983). This human isolate has been used previously by our group in numerous in vitro experiments and a rabbit study involving ureteral stents and has shown to persist and induce an inflammatory response similar to a human urinary tract infection (Pechey et al., 2009).

1.10.2.1 Identifying The Ideal Inoculum To Use In The Rabbit Study

To identify the ideal inoculum to use to induce a urinary tract infection in the animals of the rabbit study, we performed a pilot study on 6 rabbits. All of the rabbits were

catheterized with an uncoated polyurethane catheter as described in (3.1.1). Half of the rabbits were inoculated with 10E7 of E. coli GR 12 and the other half was inoculated with 10E8 of E. coli GR12. Urine samples were collected at day zero, one, three, five and seven under anesthesia as described in (3.1.2). On day 7, the animals were sacrificed after anesthesia and the urinary catheters were collected. Data on urinary bacterial counts and bacterial attachment were quantified as described in 3.3.1 and 3.3.2. The results of this pilot study indicated a bacterial inoculum with 10E8 of E. coli to be more successful and

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consistent at inducing a urinary tract infection and bacterial attachment on the catheters than using a 10E7 inoculum of E. coli.

1.10.2.2 Identifying The Ideal Inoculum To Use In The Porcine

Study

To identify the ideal inoculum to use to induce a urinary tract infection in the animals of the porcine study, we performed a pilot study on 8 pigs. Four of these pigs were stented bilaterally with an uncoated polyurethane stent whereas the other four pigs were not stented. The procedure was performed as described in (3.2.1). Half of the pigs of each group were inoculated with 10E6 of Escherichia coli GR 12 and the other half was inoculated with 10E7 of E. coli GR12. Urine samples were collected at day zero, one, three and seven under anesthesia as described in (3.2.2). On day 7, the animals were sacrificed after anesthesia. All animals in the 10E7 group had a positive urine culture on day 7 whereas only 2 animals had a positive urine culture at endpoint in the 10E6 group. It should be mentioned that the two animals having a positive urine sample at day 7 in the 10E6 group were stented. The amount of bacterial attachment on the stents was similar in both groups. Bacterial attachment was quantified as described in (3.2.3). The results of this pilot study demonstrated a bacterial inoculum with 10E7 of E. coli to be more successful at inducing a urinary tract infection than using a 10E6 of E. coli.

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Chapter 2

2

Materials and Methods

The study was approved by the University of Western Ontario animal use subcommittee (Appendix 1). As mentioned previously, the novel coatings evaluated in this project were developed based on the results of prior research and noted to contain a new DOPA- anchored long chain polymer backbone with the addition of silver nitrate and quaternary amines. Two different coatings (henceforth referred to as coatings A and B) were tested in this experiment, both containing a different amount of S-095. The test devices were coated with the two experimental coatings by DSM (DSM Biomedical, Exton, PA, USA), sterilized and packaged separately.

Our objective was to evaluate whether or not these new coatings containing silver and quaternary amines would be as effective in an in vivo model. As the rabbit model had previously been shown to be suitable for preliminary testing of urinary devices (Cadieux et al., 2006; Morck et al., 1994, 1993; Multanen et al., 2002; Olson et al., 1989), it was selected for our initial in vivo work . To test ureteral stents coated with our experimental coating compound, we chose a porcine model for our in vivo experiments. The porcine model provides a urinary tract that resembles the macroscopic human urinary tract fairly well and is a very accessible animal for laboratory testing.

All animals were allowed an acclimatization period of at least three days in the animal testing facility after arrival, prior to experimentation. Laboratory feed and drinking water were provided ad libitum during their entire stay, including before and during

experimentation.

The animal subjects were provided with environmental enrichment in accordance to species-specific standard operating protocol (SOP). The temperature and humidity of the housing space was monitored daily, and animals were provided with a cycle of 12 hours of light followed by 12 hours of darkness.

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