Biomaterials-Related Infection Group, Division of Orthopaedic and Accident Surgery, C Floor West Block, University Hospital, Nottingham NG7 2UH, UK
Received 15 August 2003; returned 12 November 2003; revised 12 January 2004; accepted 13 February 2004
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Abstract |
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Methods: Plain and impregnated material, with and without plasma protein conditioning film, were exposed to Staphylococcus epidermidis. Bacterial adherence was determined by scanning electron microscopy, chemiluminescence and culture. Time taken to kill 100% of bacteria (tK100) was determined by serial chemiluminescence, culture and the use of differential viability microscopy.
Results: The antimicrobial material did not reduce bacterial adherence. However, 100% of attached bacteria were killed in 4852 h, even in the presence of a conditioning film.
Conclusions: Impregnated antimicrobial material is likely to reduce shunt infection rates significantly without the risks and side effects of systemic antibiotics.
Keywords: hydrocephalus shunt infections, prevention, catheters
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Introduction |
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In view of this difficulty in bringing about a reduction in the high infection rate, we developed a process whereby a shunt catheter could be impregnated with antimicrobials after manufacture.10 We later showed that such catheters could resist bacterial colonization for up to 50 days in a stringent simulation model, even in the presence of a plasma protein conditioning film and under flow conditions.11 We then set out to determine how the antimicrobial catheters were able to exert their effect, and whether they inhibited bacterial adherence or killed adhered bacteria, and if so, on what time scale.
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Materials and methods |
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Medical-grade barium-filled silicone elastomer sheet identical to the material used in shunt catheters and processed as described previously8 was donated by Codman and Shurtleff Inc. (Raynham, MA, USA). Briefly, the process consisted of expansion of the silicone matrix with chloroform in which the antimicrobial(s), in this case rifampicin and clindamycin hydrochloride, were dissolved, both at 2% w/v. The chloroform was then removed by evaporation leaving molecules of the antimicrobials distributed throughout the whole of the silicone, which was then sterilized by autoclaving. Discs of the processed sheet and unprocessed controls, 4.8 mm diameter and 0.45 mm thick, were cut aseptically. For application of the conditioning film, one series of discs was exposed to 50% fresh human plasma (National Blood Service, Sheffield, UK) at 37°C, with rocking for 1 h, then rinsed in PBS.
Determination of bacterial adherence
Both series of discs and unprocessed controls (with and without plasma) were then exposed for 1 h to a suspension of Staphylococcus epidermidis, APIStaph profile 6706113 (BioMérieux, Basingstoke, UK), isolated from a CSF shunt infection. For this purpose, the bacteria were grown to mid log phase on a shaker at 37°C for 5 h, washed once in PBS and adjusted to OD490 0.6 (107 cfu/mL). After exposure to the bacterial suspension, the discs were gently rinsed in PBS. Three discs of each series were then sonicated (Ultrawave Ltd, Cardiff, UK) for 20 min at 50 Hz and the sonicates examined by chemiluminescence to determine the numbers of adhered bacteria. Briefly, 125 µL of sonicate was added to each of three wells of an opaque 96-well tray (Zeptogen Ltd, Middlesex, UK), and 125 µL of 2% tryptone soya broth (TSB) was added to three additional wells as a blank. One hundred and twenty-five microlitres of lysing agent (Bactolyse, Lumitech Ltd, Nottingham, UK) was then added to all wells and allowed to stand for 10 min at room temperature. The tray was then read in a luminometer (MicroLumat Plus LB96V, Berthold Technologies GmbH, Bad Wildbad, Germany). The assay detects bacterial ATP, present only in living cells. The chemiluminescence assay was calibrated by plate counting of the planktonic culture. Sonicates were also cultured semi-quantitatively.
Determination of time to kill
Triplets of discs were rinsed and sonicated at intervals and the sonicates analysed as above to determine the time taken to kill all the attached bacteria (tK100). The culture medium (2% TSB; Oxoid, Basingstoke, UK) was changed daily. Use of 2% TSB was adopted after investigation, to ensure that bacteria survived but did not multiply during the experiments, which lasted 3 days. Survival of the bacteria in 2% TSB for the duration of the experiments was confirmed by chemiluminescence and culture. Separate triplets of discs were retrieved at the same times and the attached bacteria examined microscopically in situ after staining with Baclight differential fluorescence viability stain (Molecular Probes Europe BV, Leiden, The Netherlands). Further discs were removed after 1 h exposure to the bacterial suspension, fixed in cold acetone and examined by scanning electron microscopy (SEM).
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Results |
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Discussion |
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Hampl et al.21 have described a rifampicin-impregnated shunt catheter, produced using the same process as that used here, but containing 9% w/w rifampicin. Crystals of the drug were demonstrated on the outer surface. The catheters in our study contained 0.054% and 0.1% rifampicin and clindamycin, respectively.22 The results shown here demonstrate that such very high concentrations of drug are not necessary to achieve tK100. Moreover, we have shown that concentrations >2% w/w result in deteriorating mechanical properties of the silicone.13 In addition, the use of rifampicin alone is likely to select for resistant mutants.23 The dual-drug principle applied in the catheters in our study, in which simultaneous mutations in genes coding for completely different targets (RNA polymerase and ribosomal binding site) are required for survival, is intended to make it extremely unlikely that resistant mutants would appear.23 The bacterial challenge used in the investigations reported here is high, and this would make detection and selection of rifampicin-resistant mutants more likely,24 but none was found.10,11 The microbial insult to the shunt is confined to the time of surgery, and no bacteria survive in the impregnated catheters. In addition, antimicrobial activity is confined to the Nernst layer25 at the shunt surface, and is dissipated rapidly beyond this. The antimicrobials are therefore unable to impact on the normal flora sites to select resistant strains. Systemically administered prophylactic antibiotics affect the patient and all the normal flora sites, resulting in side effects and risk of resistance, in the case of shunting without clinical benefit. We therefore consider that the use of silicone shunt catheters impregnated with rifampicin and clindamycin is likely to bring about a significant reduction in shunt infection, with minimal risk of side effects or development of resistance.
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Acknowledgements |
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Footnotes |
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References |
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