Principle of the In Vitro Model

All silicone Foley catheters were used in either their “as received” state for control purposes or impregnated with rifampicin, triclosan and sparfloxacin as described in Section 2.2.4. Section 3.2.2.1 to 3.2.2.3 detail the in - vitro model set, assembly, testing and sampling procedure and Figure 3.3 depicts this. TSB was used as the perfusion medium at a flow rate of 0.5 mL/min. The lumens of the urinary catheters were inoculated with 105 cfu/mL of bacteria suspended in TSB (E.coli, E.faecalis, K.pneumoniae, MRSA, P.mirabilis) and catheter ends clamped for 1 hour to allow the bacteria in the suspension to attach to the luminal surface. The tK100 screening tests and previous studies have indicated that 1 hour exposure time is sufficient for bacteria to become attached to material [81, 174]. The flow was restarted and samples of perfused TSB taken from the distal end of the catheters were collected on a daily/near to daily basis. 200 µL was plated out onto SBA or CLED and incubated for up to 72 hours. Surviving colonies were counted. If no bacterial counts were detected, catheters were re - challenged on a weekly basis and new control catheters set up. Water surrounding the catheter in the glass tube was replaced on a weekly basis to prevent the build - up of antimicrobial agents. Tests were carried out at 37°C in triplicate. Challenges were made for up to 12 weeks or until the antimicrobial catheters failed to stop bacterial colonisation. Bacteria isolated from colonised antimicrobial catheters were identified and the MIC of rifampicin, sparfloxacin and triclosan determined as detailed in Section 2.2.3 and Appendix 1. All test catheters and a colonised control catheter from each of the bacterial species were subject to evaluation by Scanning Electron Microscopy (SEM).

109 Inoculation point Water jacket Catheter 1 x 3mm tubing Parafilm Pump tubing Silicone Bung

Flask containing Sample Point 2 x 4mm tubing

perfusion medium (TSB)

Figure 3.3: In - vitro model.

Waste vessel decontaminated with Trigene Glass connector Heating circuit set at 37°C Pump

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3.2.2.1. Description of In - Vitro Model Set - Up

Part 1: Perfusion Fluid Reservoir

Step Action

1 2 L flasks of TSB were made up as per manufacturer‟s instructions 2 A 3 mm hole was made through the bung/lid of the flask and 1 x 3

mm tubing (SLS, Nottingham, UK) thread through until it reached the bottom of the fluid

3 A glass connector (1 mm diameter) was inserted into the other end of the 1 x 3 mm tubing

4 A clamp was placed onto the tubing to ensure the fluid does not escape

5 The top of the flask and the end of the tubing was covered with aluminium foil and secured with tape

6 All was sterilised by autoclaving. TSB was renewed as necessary

Part 2: Inlet Tubing Step Action

1 A glass connector was pushed into one end of the pump tubing 2 Attached to the above glass connector was 1 x 3 mm tubing

3 A short piece of 2 x 4 mm tubing (SLS, Nottingham, UK) was attached to the other end of the 1 x 3 mm tubing

4 A straight connector 3 - 5 mm (SLS, Nottingham, UK) was connected to the 2 x 4 mm tubing

5 The end of the connectors were covered with aluminium foil and sterilised by autoclaving

Part 3: Catheter Section Step Action

1 A suitable sized hole was made through the 14 x 20 mm silicone stoppers (SLS, Nottingham, UK) that fit into the glass tubes within the water jacket

2 The control and antimicrobial catheters were cut to size (minus both ends of the catheter) and pushed through the hole in the silicone stopper until approx 2.5 cm poked through each end

111 Part 4 : Outlet Tubing

Step Action

1 To one end of a straight connector a small section of 2 x 4 mm tubing was attached

2 1 x 3 mm tubing was inserted into the end of the 2 x 4 mm tubing 3 Ends of the tubing and connector were covered with aluminium foil

and sterilised by autoclaving

Part 5: Waste Collection Vessel Step Action

1 A 30 L Sharps Container was used for the collection of waste perfusion medium (TSB)

2 Disinfectant (Trigene) was added to kill bacteria within waste perfusion fluid

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3.2.2.2. Assembly of In - Vitro Model

Step Action

1 The water jacket was filled with distilled water and maintained at 37°C by the heating circuit

2 From Part 2 of the inlet section, the pump tubing was clipped into place on the pump

3 The 1 x 3 mm tubing from Part 1 of the perfusion reservoir was connected to the glass connector of the pump tubing

4 The catheter from Part 3 was inserted up the centre of the tubes in the water jacket and the silicone stopper pushed securely into the bottom of the glass tubes

5 The glass tubes of the water jacket were filled with distilled water to 2 cm from the top and Parafilm wrapped around the top where the catheter protruded

6 The straight connector from Part 2 of the inlet tubing was fitted into the top of the catheter

7 To the bottom of the catheter, the straight connector from Part 4 of the outlet tubing was pushed through

8 The end of the 1 x 3 mm tubing from Part 4 of the outlet tubing was inserted into the waste collection vessel. The tubing was held in place with tape to ensure that it did not touch the sides of the container. For each bacterial strain separate waste containers for both the antimicrobial and control catheters were used

9 The clamp from the tubing attached the perfusion reservoir in Part 1 was removed and the pump started at a flow rate of 0.5 mL/min. Checks were made to ensure that no leaks occurred and that the TSB flowed through unimpeded

10 The system was allowed 1 hour to stabilize and reach 37°C before the catheters were challenged with bacteria

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3.2.2.3. Testing and Sampling Procedure

Several colonies of MRSA, E.coli, E.faecalis, K.pneumoniae, or P.mirabilis from overnight SBA or CLED plates were placed into separate universal containers of 20 mL of TSB. These were incubated in a 37°C orbital shaker set at 200 rpm for 4 hours and grown to early log phase. Cultures were standardised to achieve an optical density (490 nm) of between 0.6 - 0.7 using a Jenway 6705 UV/VIS spectrophotometer. This gives approximately 108 cfu/mL which was diluted in TSB to 105 cfu/mL and inoculated down the lumens of the catheters.

Inoculation Procedure Step Action

1 A clamp was placed onto the inlet tubing and the pump turned off

2 The straight connector on the top of the catheter was removed and the tip of the catheter swabbed using a steret

3 A 2 mL syringe was filled with the bacterial suspension and injected down the catheter lumen trying to avoid any air bubbles

4 Once the bacterial suspension reached the distal end of the catheter, the outlet tubing was clamped

5 The area around the inoculation site was swabbed with a steret and the connector replaced

6 The system was left for 1 hour to allow the bacteria to attach to the catheter surface

7 After 1 hour, the clamp attached to the outlet tubing was removed followed by the clamp attached to the inlet tubing

8 The pump was restarted and the perfusion medium flowed through for approximately 1 hour

9 The inlet tubing with connector section and the outlet tubing with connector section were then renewed to prevent the migration of bacteria that may have attached to the silicone tubing from travelling to the test catheter

114 Sampling Procedure

Step Action

1 The outlet tubing (Part 4) connected from the distal end of the urinary catheter was disconnected for sampling

2 A 7 mL bijou bottle was placed under the end of the catheter and approximately 1 mL of the perfusion medium running through the catheter collected

3 The end of the catheter and outlet tubing connector were swabbed with a steret before rejoining and the system monitored to ensure no leaks occurred

4 The collected effluent was vortexed and 200 µL plated onto SBA or CLED. If the sample showed turbidity, 1:10 dilutions were made as appropriate

5 Plates were incubated at 37°C for up to 72 hours

6 If growth was detected, bacterial counts were recorded as cfu/mL 7 If there was no growth, plates were re - incubated for a further 24 - 48

hours

8 Samples were collected on or as near to a daily basis as possible

9 If no bacterial growth was shown following day 6 of sampling, the inoculation procedure was repeated each week (every 7 days) until bacterial colonisation persisted or until the 12 week point was reached. 10 Following successful colonisation of control catheters, new ones were

set up each week

11 If bacterial growth persisted in the antimicrobial catheter, the test was stopped and the catheter taken down

12 The identity of the bacteria isolated from the colonised antimicrobial catheters was checked and MICs of rifampicin, triclosan and sparfloxacin determined

13 At the end of the test, approximately 5 x 1 cm segments were cut from all catheters and stored in cold acetone for Scanning Electron Microscopy (SEM) analysis

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