Antibacterial activity of antibiotic-soaked polyvinylpyrrolidone-grafted silicon elastomer hydrocephalus shunts

Jaap J. Boelensa,*, Wee-Fu Tanb, Jacob Dankerta and Sebastian A. J. Zaata

a Department of Medical Microbiology and b Department of Neurosurgery, Academic Medical Center, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
If shunts, inserted for the relief of hydrocephalus, are pretreated with antimicrobials, the incidence of shunt-associated infections (SAI) may be reduced. The duration of the antibacterial activity of shunts, made from conventional silicon elastomer (SE) or from SE grafted with the hydrogel polyvinylpyrrolidone (SEpvp), which had been soaked in various antibiotics, was assessed in vitro. For any antibiotic or combination, using an arbitrary breakpoint (aBP), SEpvp remained antibacterially active for longer periods than SE. Bacterial adherence to either shunt was prevented during the period of antibacterial activity. Thus, the aBP is a good indicator of the capacity of antimicrobial-treated shunts to prevent bacterial colonization in vitro. Hydrogel-grafting of shunts may be useful in preventing SAI.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Infection of a shunt, implanted for the relief of hydrocephalus, is a major problem in neurosurgery. The average incidence of shunt-associated infections (SAIs) is approximately 10%. SAI is predominantly caused by Staphylococcus epidermidis (40–75%).1

The initial step in the pathogenesis of SAI is assumed to be adherence of bacteria.1 To prevent infection, a surface-modified silicon elastomer, the polyvinylpyrrolidone-grafted silicon elastomer (SEpvp) was introduced. As polyvinylpyrrolidone is a hydrogel, SEpvp may take up more antibiotic when soaked in an antibiotic solution than conventional silicon elastomer (SE) shunts,2 with resulting longer antibacterial activity and prevention of bacterial adherence. This study examines the in vitro antibacterial activity of SEpvp and SE soaked in solutions of various antibiotics or combinations and correlations with bacterial adherence.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotics

Solutions of antibiotics, containing maximum soluble concentrations, were prepared in sterile distilled water.3 The antibiotics used were: gentamicin (600 mg/mL; Centrafarm, Etten-Leur, The Netherlands), teicoplanin (135 mg/mL; Yamanouchi, Leiderdorp, The Netherlands), minocycline (100 mg/mL; Lederle, Hoofddorp, The Netherlands), flucloxacillin (650 mg/mL; SmithKline Beecham, Rijswijk, The Netherlands) and vancomycin (200 mg/mL; Eli Lilly, Nieuwegein, The Netherlands). Seven serial two-fold dilutions, of each of these antibiotic solutions, were prepared. Rifampicin (60 mg/mL; Yamanouchi) was dissolved and serial two-fold dilutions were made in the solvent provided by the manufacturer (water and polysorbate 81).

Preparation of shunt segments

Shunts of SE and SEpvp (Bioglide), with a diameter of 2.5 mm and a wall thickness of 0.6 mm, were obtained from Medtronic PS Medical, Goleta, CA, USA. Segments, cut under aseptic conditions, were soaked in the antibiotic solutions for 15 min, rinsed twice with phosphate buffered saline (PBS) and dried in a laminar flow cabinet for 10 min. In all experiments saline-soaked SE and SEpvp were used as controls.

Bacterial strains

Two clinical slime-producing isolates, S. epidermidis AMC1 and Staphylococcus capitis AMC2 were used.4 Strain AMC1 was highly susceptible to rifampicin, teicoplanin, minocycline and vancomycin, and strain AMC2 to rifampicin, gentamicin and flucloxacillin. Strain AMC1 was used to determine the antibacterial activity of SE and SEpvp soaked in rifampicin, teicoplanin, minocycline, vancomycin, rifampicin+teicoplanin, rifampicin+minocycline and rifampicin+vancomycin. Strain AMC2 was used to determine the antibacterial activity of SE and SEpvp soaked in gentamicin, flucloxacillin and rifampicin+gentamicin.

Preincubation of antibiotic-soaked shunt segments in agar, serum or subcutaneously

Using a pair of tweezers, 5 mm long antibiotic-soaked (ab)-SE and ab-SEpvp segments were placed into 2.5 mm diameter holes, punched in a 5 mm thick 1% (w/v) IsoSensitest agar plate (Oxoid, Basingstoke, UK). In addition, rifampicin-soaked segments were immersed in 40 mL human serum (60 mL bottles) and placed on a rotary-shaker (30 rpm; 37°C) or were implanted in rabbit subcutaneous tissue (four New Zealand Whites; 2- to 3-months-old and weighing 2500–3000 g; Harlan, Horst, The Netherlands). Segments were removed from either environment after 0, 3, 9, 24, 48, 72, 96 or 120 h and tested for antibacterial activity.

Antibacterial activity of antibiotic-soaked shunt segments

The antibacterial activity of the segments was assessed by an agar diffusion test on Iso-Sensitest agar plates (15 cm; Oxoid). The bacterial inoculum was prepared according to NCCLS standards (Kirby-Bauer).5 Sets of five SE and SEpvp segments, soaked in the same antibiotic solution and retrieved from the same preincubation environment, were incubated overnight (37°C) on one plate. Inhibition zones were measured in mm (mean ± S.D. mm). Antibacterial activities of SE and SEpvp, based on arbitrary zone breakpoints (aBP), were compared. The duration of high antibacterial activity is defined as the period of preincubation in either environment, after which the segments still produce inhibition zones equal to or larger than the aBP. The aBP derived from the NCCLS zone breakpoint for susceptibility of coagulase-negative staphylococci using the disc diffusion method,5 was corrected for the difference in diameter between antibiotic discs (9 mm; r = 4.5 mm) and shunts (2.5 mm; r = 1.25 mm). For example the NCCLS BP for rifampicin discs is 23 mm (r = 11.5 mm), and the calculated aBP for rifampicin-soaked shunts is 21.4 mm (10.7 mm). This aBP is calculated as follows, {pi}. (r aBP)2 = {pi}. (11.5)2 {pi}. (4.5)2 + {pi}. (1.25)2 = 356.8. Consequently, the r for the aBP is 10.7 mm, and the diameter is 21.4 mm. The aBP diameters for rifampicin, flucloxacillin, gentamicin and minocycline were 21.4 mm, and for vancomycin and teicoplanin 12 mm.

Bacterial adherence to ab-SE and ab-SEpvp

An inoculum of 108 cfu/mL Trypticase–Soy Broth (TSB; Difco, Detroit, MI, USA) was prepared. TSB was used to mimic a protein-rich environment. After various preincubation periods in agar, 10 cm long SE and SEpvp segments were exposed to the inoculum in 100 mL TSB for 3 h (37°C). Subsequently, segments were rinsed twice with PBS, the middle 3 cm was sonicated in a tube containing 3 mL of PBS (Bransonic B-2200 E4, 47 kHz, 205 W, 30 s) to dislodge the adherent bacteria. The 3 mL of PBS was quantitatively cultured (mean ± S.D. cfu/cm). Statistical analysis was performed by Student's t test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The antibacterial activity of both shunts showed a high initial decline during the first 3–9 h, followed by a more gradual decrease. For each antibiotic, SEpvp had longer high antibacterial activity than SE. Shunts soaked in solutions containing the maximum soluble antibiotic concentrations had the longest duration of high antibacterial activity. Table IGo shows the estimated duration of high antibacterial activity of shunts soaked in the maximum soluble concentration. SE and SEpvp soaked in the most dilute solutions (1:64) of the antibiotics had no high antibacterial activity, except for the segments soaked in rifampicin (2 h and 24 h), teicoplanin (0 h and 2 h) or vancomycin (1 h and 1 h). The only combination which demonstrated greater activity than single antimicrobials was SEpvp soaked in the combination of rifampicin+ teicoplanin, which showed an increased period of high antibacterial activity (84 h) compared with either rifampicin or teicoplanin alone.


View this table:
[in this window]
[in a new window]
 
Table I. Estimated periods of high antibacterial activity (zone of inhibition >aBP; see Materials and methods) and timepoints of first bacterial adherence to SE and SEpvp soaked in antibiotic solutions of maximum soluble concentrationa
 
For the segments of SE and SEpvp with zones of inhibition larger than the aBP, no bacterial adherence occurred (Table IGo). The adherence of bacteria to saline-soaked SEpvp was significantly less (P < 0.001) than on saline-soaked SE (Table IIGo). Bacterial adherence to ab-SEpvp was significantly less and occurred significantly later for all the antibiotics tested compared with SE (Table IIGo).


View this table:
[in this window]
[in a new window]
 
Table II. Number of cfu/cm of S. epidermidis AMC1 or S. capitis AMC2 adherent to SE and SEpvp. Segments soaked in solutions of maximal soluble concentration of various antibiotics, preincubated in agar for different periods, removed and subsequently placed in TSB containing 108 cfu/mL for 3 h
 
Rifampicin-soaked SE and SEpvp removed from agar and from rabbit subcutaneous tissue had similar durations of high antibacterial activity. By contrast, SE and SEpvp immersed in serum had less antibacterial activity, namely 4 h and 12 h, respectively.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study demonstrates that: (i) grafting of SE with the hydrogel polyvinylpyrrolidone enhances retention of antimicrobial activity, (ii) the aBP can be used to predict bacterial colonization in vitro and (iii) agar is a good experimental model in the study of the antibacterial activity of rifampicin-soaked shunts, implanted subcutaneously.

Grafting of SE with polyvinylpyrrolidone prolonged the antibacterial activity of ab-SEpvp shunts compared with ab-SE. Sherertz et al.6 reported that chlorhexidine-coated catheters producing zones of inhibition with a diameter of >15 mm for Staphylococcus aureus prevented subcutaneous catheter-associated infection in rabbits. This 15 mm zone size has been used as a breakpoint to predict the in vivo efficacy of antimicrobial shunts, regardless of the antibiotic and the test organism used.7 The aBP, derived from the NCCLS breakpoints for antibiotic susceptibility, taking bacterial species and antibiotics used into account, seems to be a good indicator in predicting in vitro bacterial adherence to antibacterially active shunts.

The advantages of hydrogel-coated shunts are that clinicians can choose a specific antibiotic or antibiotic combination and that the antibiotics will be released from the shunt for up to 4–5 days. It should be noted that the duration of antibacterial activity in vivo in the proximal end (ventricle), and in the lumen of the shunt could be shorter, because of the flow of CSF. Although much longer periods of in vitro antibiotic release of shunts coated with antibiotics have been reported,8–10 such shunts can have disadvantages. If a patient is allergic to the antibiotic in or on the shunt, or if local resistance rates to the antibiotic coated on to the shunt are high, then the shunt cannot be used. The risk of the emergence of resistance to the antibiotic on the shunt must be considered, since subinhibitory antibiotic concentration in the antibiotic gradient around the implanted shunt could be present for long periods. Soaking of SEpvp shunts in the maximum soluble antibiotic concentration should not lead to toxic effects. The inhibition zones obtained are smaller than those that correlate with concentrations of antibiotic with which toxicity could be associated.7 In addition, the inflammatory tissue reaction around ab-SE and ab-SEpvp in rabbit subcutaneous tissue and around saline-soaked segments were similar (data not shown).

Since it is assumed that SAIs originate from contamination during insertion of the catheter or shortly thereafter,1 antimicrobial-treated shunts should release antibiotics during the first few days after implantation. Therefore, the use of the SEpvp shunt may contribute to the prevention of SAI.


    Acknowledgments
 
The studies were approved by the Institutional Animal Care and Use Committee of the Academic Medical Center, University of Amsterdam, The Netherlands. Part of these data was presented at the Thirty-Seventh Interscience Conference on Antimicrobial Agents and Chemotherapy 1997, Toronto, Canada, Abstract J-111.


    Notes
 
* Correspondence address. Department of Pediatrics, Leiden University Medical Centre, J6-S, PO Box 9600, 2300 RC Leiden, The Netherlands. Tel: +31-71-5262824; Fax: +31-71-5248198; E-mail: BoelensJJ{at}yahoo.com

Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Seifert, H., Jansen, B. & Farr, B. M. (1997). Catheter-Related Infections. Marcel Dekker, New York.

2 . Giammona, G., Pitarresi, G., Tomarchio, V., Cacciaguerra, S. & Govoni, P. (1997). A hydrogel based on a polyaspartamide: characterization and evolution of in-vivo biocompatibility and drug release in the rat. Journal of Pharmacy and Pharmacology 49, 1051–6.[ISI][Medline]

3 . Reynolds, J. E. F. (1989). Martindale: the Extra Pharmacopoeia, 29th edn. The Pharmaceutical Press, London.

4 . Christensen, G. D., Simpson, W. A., Bisno, A. L. & Beachey, E. H. (1982). Adherence of slime-producing strains of Staphylococcus epidermidis to smooth surfaces. Infection and Immunity 37, 318–26.[ISI][Medline]

5 . National Committee for Clinical Laboratory Standards. (1997). Performance Standards for Antimicrobial Disk Susceptibility TestsSixth Edition: Approved Standard M2-A6. NCCLS, Wayne, PA.

6 . Sherertz, R. J., Carruth, W. A., Hampton, A. A., Byron, M. P. & Solomon, D. D. (1993). Efficacy of antibiotic-coated catheters in preventing subcutaneous Staphylococcus aureus infection in rabbits. Journal of Infectious Diseases 167, 98–106.[ISI][Medline]

7 . Raad, I., Darouiche, R., Hachem, R., Mansouri, M. & Bodey, G. P. (1996). The broad-spectrum activity and efficacy of catheters coated with minocycline and rifampin. Journal of Infectious Diseases 173, 418–24.[ISI][Medline]

8 . Bayston, R. & Lambert, E. (1997). Duration of protective activity of cerebrospinal fluid shunt catheters impregnated with antimicrobial agents to prevent bacterial catheter-related infection. Journal of Neurosurgery 87, 247–51.[ISI][Medline]

9 . Schierholz, J., Jansen, B., Jaenicke, L. & Pulverer, G. (1994). In-vitro efficacy of an antibiotic releasing silicone ventricle catheter to prevent shunt infection. Biomaterials 15, 996–1000.[ISI][Medline]

10 . Schierholz, J. M. & Pulverer, G. (1997). Development of a new CSF-shunt with sustained release of an antimicrobial broad-spectrum combination. Zentralblatt fur Bacteriologie 286, 107–23.

Received 10 March 1999; returned 28 July 1999; revised 26 August 1999; accepted 18 October 1999