Triclosan Loaded Ureteral Stents Decrease Proteus Mirabilis 296 Infection in a Rabbit Urinary Tract Infection Model

Triclosan Loaded Ureteral Stents Decrease Proteus Mirabilis 296 Infection in a Rabbit Urinary Tract Infection Model Peter A. Cadieux,* Ben H. Chew,*,† Bodo E. Knudsen,‡ Kathy DeJong, Elaine Rowe, Gregor Reid and John D. Denstedt§,储 From the Departments of Surgery (Division of Urology) (BHC, BEK, GR, JDD), Microbiology and Immunology (PAC, GR), Chemistry (KD) and Pathology (ER), University of Western Ontario and Canadian R and D Centre for Probiotics (PAC, BHC, BEK, GR, JDD), London, Ontario, Canada

Purpose: Infection and encrustation remain major limitations of ureteral stent use and to our knowledge no device has completely overcome these obstacles to date. Triclosan is a biocide currently used in a plethora of consumer and medical products that has recently been loaded into a ureteral stent. Using a rabbit model of UTI we examined the effects of triclosan impregnated stent segments on the growth and survival of Proteus mirabilis, a uropathogen commonly associated with device related UTI and encrustation. Materials and Methods: A total of 48 male New Zealand White rabbits were instilled transurethrally with 1 ⫻ 106 P. mirabilis 296. A stent curl from a triclosan eluting, Percuflex Plus® or Optima® ureteral stent was placed intravesically. Urine was cultured on days 1, 3 and 7. On day 7 the stents were assessed for encrustation and viable organisms, while the bladders were scored for the degree of inflammation. Results: Throughout the study urine isolated from the triclosan group contained significantly fewer viable organisms than controls with 7 of 13 animals completely clearing the infection by day 7. Similarly 9 of 13 triclosan eluting stents showed no viable organisms upon recovery and the remaining 4 showed significantly fewer organisms than controls. Urine and stents in all controls were positive for P. mirabilis at all time points. Although there was no significant difference in encrustation among the groups, bladders harvested from the triclosan group demonstrated significantly less inflammation. Conclusions: Triclosan eluting stents greatly decreased P. mirabilis growth and survival in a rabbit UTI model compared to controls. These stents may prove useful for decreasing device related P. mirabilis UTIs. Key Words: ureter, stents, urinary tract infections, triclosan, rabbits

reteral stents are used commonly in the urological armamentarium for various reasons but their longterm and widespread use is limited by infection and encrustation. Urinary prostheses are constantly exposed to urine and they develop a film of urinary constituents within minutes of placement. This creates a platform for bacterial adherence, colonization and infection.1 Ureteral stents removed from patients frequently show adherent bacteria and along with potential bacterial spillage during surgical procedures this has led to the routine use of oral antibiotics at stent placement.1 While this has decreased infection rates postoperatively, stent associated UTIs remain a significant

U

Submitted for publication July 6, 2005. Study received university Animal Care and Use Committee approval. Supported by an unrestricted grant from Boston Scientific Corp. and a Canada Graduate Scholarship (Canadian Institutes of Health Research) (PC). * Equal study contribution. † Financial interest and/or other relationship with Boston Scientific, ACMI and Cook Urological. ‡ Financial interest and/or other relationship with Boston Scientific. § Financial interest and/or other relationship with Boston Scientific, Cook Urological and Olympus. 储 Correspondence: St. Joseph’s Health Care, 268 Grosvenor St., London, Ontario N6A 4V2, Canada (telephone: 519-646-6036; FAX: 519-646-6037; e-mail: [email protected]).

0022-5347/06/1756-2331/0 THE JOURNAL OF UROLOGY® Copyright © 2006 by AMERICAN UROLOGICAL ASSOCIATION

problem.2 This process occurs on all currently commercially available stents and catheters,3 and it can lead to significant patient morbidity.2 As a means of survival in the urinary tract several uropathogenic species of bacteria metabolize urea, a major component of the urinary milieu. They accomplish this through the expression of urease, an enzyme that reduces urea to ammonium. One such species is Proteus mirabilis, a gramnegative organism commonly associated with urinary tract infections. As increasing levels of ammonium build up in urine during infection with urease producing pathogens, urinary pH is increased, resulting in the precipitation of calcium and magnesium phosphates. These precipitates can form infection (struvite) stones and/or adhere to urinary devices as encrustation. Additionally, like most uropathogens these organisms can form biofilms that resist oral antibiotic treatment, making device removal necessary to treat the infection. New stent biomaterials and coatings have been created in an attempt to decrease biofilm formation, encrustation and infection but to our knowledge no ideal material or coating exists to date. Triclosan is a potent, broad-spectrum antimicrobial currently used in numerous products, including mouthwashes, toothpastes, surgical scrubs and more recently polyglactin suture.4 – 6 It acts as a specific binding inhibitor of the bacterial enoyl-acyl carrier protein reductase, leading to a

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Vol. 175, 2331-2335, June 2006 Printed in U.S.A. DOI:10.1016/S0022-5347(06)00252-7

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TRICLOSAN LOADED URETERAL STENTS AND URINARY TRACT INFECTION

blockage of fatty acid biosynthesis and ultimately destabilization of the cell membrane. In addition to its effective biocidal activity, triclosan also has an outstanding safety record as well as proven anti-inflammatory properties.7 Previous work has shown that Foley catheter balloons inflated with triclosan inhibited biofilm formation and encrustation deposition in an in vitro model of Proteus mirabilis bladder infection.8 To our knowledge a triclosan eluting urinary prosthetic has not been described in vivo. We hypothesized that triclosan eluting ureteral stents would decrease bacterial growth in urine and lower bacterial deposition on the device compared to 2 commercially available stents. As a result, we hypothesized that less stent encrustation and bladder inflammation would also ensue.

TABLE 1. Grading system for assessing histological bladder inflammation13 Grade

Description

0 1 2

Less than 15 polymorphonuclear cells/hpf, 0.44 mm diameter 15–50 cells/hpf 50–50 cells/hpf, relatively loose infiltrate without distortion of underlying tissue Greater than 300 cells/hpf, density sufficient to obscure underlying tissue architecture Necrosis

3 4

for inflammation using the histological grading system previously described by Watterson et al (table 1).12 Data were compared using the 2-tailed Student t test with p ⬍0.05 considered statistically significant.

MATERIALS AND METHODS A total of 48 male New Zealand White rabbits were randomized to intravesical placement of the pigtail portion of a Percuflex Plus®, Optima® or triclosan loaded (Triumph®) stent. All animal protocols were approved by the university Animal Care and Use Committee. General anesthesia was achieved using an intramuscular injection of acepromazine (0.1 mg/kg) and glycopyrrolate (0.01 mg/kg), followed by inhalational isoflurane administered by mask. The abdomen was shaved and prepared. A 5Fr angled ureteral catheter was used to catheterize the bladder and its position was confirmed by intraoperative ultrasound and urine aspiration. A polytetrafluoroethylene coated 0.035-inch guidewire was inserted through the catheter and trimmed stent curls were inserted into the bladder transurethrally, as described by Fung et al.9 The bladder was emptied, urine was cultured and 1 ⫻ 106 cfu Proteus mirabilis 296, a clinical strain isolated from a human infection (minimum inhibitory concentration 03 ␮g/ml),10 were injected in 1.5 ml saline, followed by 2.5 ml sterile saline to ensure that the entire amount was ejected from the catheter. The rabbits were then recovered and injected with the anti-inflammatory medication meloxicam (0.2 mg/kg). Urine was collected by ultrasound guided sterile suprapubic aspiration or expressed via suprapubic pressure using general anesthesia on postoperative days 1 and 3. The number of viable bacteria in urine was determined by serial dilution plating on nonswarming agar.11 Urine pH was also measured at each time point. Animals were sacrificed on day 7 and urine was obtained for culture after laparotomy and cystectomy. Stents were retrieved and sectioned to assess bacterial growth and encrustation. To assess viable stent adherent organisms half of the stent was rinsed and placed in saline, the encrustation was manually removed and crushed, and the stent and encrustation were sonicated for 20 minutes in a Bransonic® sonicator. The resulting solution was serially diluted and cultured on nonswarming agar.11 The second half of the stent was dried completely and the encrustation was weighed and expressed in mg/cm2 stent surface area. Total calcium was also quantified from 16 representative encrustation samples via atomic absorption spectrometry using a Spectra® 10 atomic spectrophotometer at a wavelength of 422.7 nm and a slit width of 0.5 nm. The encrustation was dissolved in 3 ml 40% HCl and 10,000 ppm lanthanum before analysis. The bladders were fixed in 10% formalin, sectioned for histopathology and assessed by a blinded pathologist (ER)

RESULTS Table 2 lists all numerical study data. At necropsy 35 stents successfully remained in the bladder, including 13 triclosan, 12 Percuflex® and 10 Optima® stents. The remaining stents were located in the prostatic utricle (11) and peritoneum (2), and were excluded from analysis. All rabbits recovered well from surgery and continued to feed, drink and gain weight normally. Urine culture revealed significantly less P. mirabilis in the triclosan group than in the Percuflex Plus® group at all time points and in the Optima® group on days 3 and 7 (fig. 1). There were no statistical differences between the 2 control groups at any time point. While all 22 control animals had positive urine cultures throughout the experiment (greater than 105 cfu/ml), in the triclosan group the infection cleared in 4 of 13 animals by day 1, in 5 of 13 by day 3 and in 7 of 13 by day 7. Five of the 6 animals in the triclosan group that continued to have positive urine cultures had 1 ⫻ 103 to 1 ⫻ 105 cfu/ml and the other had 1 ⫻ 108 cfu/ml. There were significantly fewer bacteria adherent to triclosan stents than to control devices (fig. 2). Nine of the 13 triclosan stents showed no viable organisms on the surface after 7 days, while all 22 control devices were colonized. Similar encrustation levels were observed on all stents after 7 days using dry weight and atomic absorption spectrometry analyses (table 2). Also, urinary pH (range 8.3 to 9.0) showed no differences among any of the groups at any time point. Histological examination revealed that bladders in the triclosan group had significantly less inflammation than those in the Percuflex Plus® group and a decreased level compared to those in the Optima® group (p ⫽ 0.01 and 0.08, respectively). There was no difference between the 2 control stents (p ⫽ 0.61). In fact, 67% of triclosan treated animals (9 of 13) had normal appearing bladders (grade 0) on histology compared to only 25% (3 of 12) and 40% (4 of 10) in the Percuflex Plus® and Optima® groups, respectively. DISCUSSION To our knowledge this is the first study to describe the effects of a triclosan eluting ureteral stent in an in vivo model. Triclosan acts by inhibiting the enoyl-acyl carrier protein reductase, a highly conserved enzyme in bacteria that is responsible for fatty acid synthesis.13 In the current study control stents became readily colonized with P. mira-

TRICLOSAN LOADED URETERAL STENTS AND URINARY TRACT INFECTION

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TABLE 2. Urine, stent and bladder pathology data Data Collected Mean urine cfu/ml ⫾ SD: Day 1 Day 3 Day 7 No. subjects No. pos urine culture:‡ Day 1 Day 3 Day 7 Mean urine pH ⫾ SD: Baseline Day 1 Day 3 Day 7 Mean day 7 stent cfu/cm2 ⫾ SD Mean encrustation/cm2 stent ⫾ SD: Mg Ca⫹⫹ ppm Mean pathology grades 0–4 ⫾ SD

Triclosan

Optima®

p Value

Percuflex Plus®

1.6 ⫻ 102 ⫾ 9.2 ⫻ 102* 4.3 ⫻ 102 ⫾ 1.3 ⫻ 103* 3.3 ⫻ 102 ⫾ 1.0 ⫻ 103* 13

9.5 ⫻ 103 ⫾ 8.6 ⫻ 102 1.9 ⫻ 107 ⫾ 1.5 ⫻ 100† 2.6 ⫻ 106 ⫾ 1.9 ⫻ 101† 10

0.29 0.008* 0.001*

2.6 ⫻ 106 ⫾ 5.2 ⫻ 101† 4.0 ⫻ 106 ⫾ 2.7 ⫻ 101† 1.0 ⫻ 107 ⫾ 6.8 ⫻ 100† 12

9 8 6

10 10 10

8.27 ⫾ 0.45 8.73 ⫾ 0.55 8.54 ⫾ 0.60 8.74 ⫾ 0.53 1.4 ⫻ 101 ⫾ 1.3 ⫻ 102*

8.30 ⫾ 0.88 8.79 ⫾ 0.80 9.03 ⫾ 0.28 8.67 ⫾ 0.39 7.8 ⫻ 105 ⫾ 2.7 ⫻ 101†

35.0 ⫾ 14.5 2,321 ⫾ 1,072 0.39 ⫾ 0.77*

36.8 ⫾ 24 1,760 ⫾ 1,310 1.10 ⫾ 1.10

p Value vs Triclosan 0.001* 0.0006* 0.001*

12 12 12 0.92 0.87 0.12 0.75 ⬍0.0001* 0.83 0.47 0.08

8.48 ⫾ 0.40 8.93 ⫾ 0.32 8.86 ⫾ 0.46 8.86 ⫾ 0.38 8.7 ⫻ 104 ⫾ 5.9 ⫻ 101†

0.22 0.36 0.19 0.54 ⬍0.0001*

33.8 ⫾ 15.0 2,190 ⫾ 2,050 1.33 ⫾ 0.99†

0.85 0.91 0.01*

There were no statistically significantly different values for Optima® vs Percuflex Plus®. * Triclosan vs control significantly different. † Statistically significant vs triclosan. ‡ No subjects had a positive urine culture at baseline.

bilis and urine culture yielded bacteria at significant levels (1 ⫻ 106 to 1 ⫻ 107 cfu/ml). However, triclosan eluting stents completely cleared bacteria from the bladder in 54% of animals within 7 days and only 31% had viable adherent organisms on the device upon retrieval. Furthermore, only 8% of animals in the triclosan group showed greater than 1 ⫻ 106 cfu/ml P. mirabilis in urine on day 7 compared to 100%

of controls. Triclosan was clearly effective during the typical duration in which human stents are implanted. Bladder inflammation was significantly less and in significantly fewer animals in the triclosan group compared to the Percuflex Plus® group and it approached significance compared to that in the Optima® group (p ⫽ 0.01 and 0.08, respectively). Intravesical Proteus alone would likely cause sub-

FIG. 1. Urine culture. Bacterial growth in triclosan group was significantly less than in Percuflex Plus® group on all days and in Optima® group on days 3 and 7. Single asterisk indicates Percuflex Plus® vs triclosan statistically significantly different. Double asterisks indicate Optima® vs triclosan statistically significantly different.

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TRICLOSAN LOADED URETERAL STENTS AND URINARY TRACT INFECTION

FIG. 2. Stent bacterial growth in triclosan group was significantly less than in 2 controls. Single asterisk indicates Percuflex Plus® vs triclosan statistically significantly different. Double asterisks indicate Optima® vs triclosan statistically significantly different.

stantial bladder inflammation, which would be compounded by the addition of a foreign body, such as a stent. None of the animals received only a stent curl or bacteria alone and, thus, the proportion of inflammation attributable to the stent or to the bacteria is unknown. This decrease in bladder inflammation in the triclosan group could have been due to the decreased level of bacteriuria in the triclosan group compared to that in controls and/or to the anti-inflammatory properties of triclosan. The hypothesis that stent encrustation would be avoided by preventing bacterial growth and survival was not supported by this study. Reasons for this may include the fact that rabbit urine in general has a high pH of 8 to 9 due to a vegetarian diet as well as high levels of urinary calcium phosphate and magnesium, which readily precipitate at this pH. The pH at which precipitation occurs varies depending on the solubility of accompanying urinary salts. These factors lead to rapid deposition of encrustation onto urinary devices in the rabbit. This rapid precipitation and device encrustation are not common in humans and, thus, the triclosan stent may well decrease bacterial induced encrustation deposition when tested in a clinical setting. The dense deposits in the rabbit model may have provided a substrate onto which the Proteus organisms bound, resulting in 6 triclosan treated animals still being found to have Proteus infection. Despite this observation, bacteria were not able to survive in the encrustation of most devices, as evidenced by negative stent cultures in 69% of the animals in the triclosan group. This implies that the leaching triclosan was able to eradicate adherent bacteria, which is something not found previously with some antibiotics.1

Patients with an encrusted triclosan stent may be less likely to have a concomitant UTI. Patients with infected stones and urinary devices do not typically respond to antibiotics alone and they often require removal of the stone or device. Triclosan stents may prevent bacterial growth directly on the stent, preventing it from becoming a nidus for infection and allowing eluted triclosan or oral antibiotics to rid the bladder of planktonic (freely floating) bacteria. Proteus UTIs were cleared by the triclosan stent alone in more than half of the cases in this study. Whether a triclosan stent has enough antimicrobial activity to clear an established UTI in a clinical situation remains to be determined. However, due to the prevention of bacterial adherence to the stent biofilm and the constant clearance of infected urine by the host it is speculated that this stent may decrease the amount of bacteria in urine and potentially clear the infection. Elution of triclosan from the stent resulted in greatly decreased bacteriuria and adherent organisms at 7 days. The prolonged elution time of triclosan may result in antimicrobial benefits for up to 3 months based on previous elution data.14 The duration of clinical effectiveness requires further evaluation. Interestingly triclosan exerted its effects despite the presence of crystal deposits, suggesting its ability to penetrate through encrustation and kill bacteria surrounding the stent as well as those attached to crystals and the stent itself.15 With respect to resistance triclosan was thought to act like a nonspecific biocide affecting membrane integrity until its target of lipid synthesis was discovered by McMurry et al.13 The enoyl-acyl carrier protein reductase enzyme in-

TRICLOSAN LOADED URETERAL STENTS AND URINARY TRACT INFECTION volved in fatty acid synthesis that is important for membrane maintenance is now known to be a major target of the compound. The fact that triclosan has a directed target site rather than a nonspecific mechanism of action makes it theoretically possible to breed resistant organisms. In fact, natural resistance to triclosan has been demonstrated and the mechanisms are similar to those noted in bacteria for other antibiotics.16 –19 There has been no resistance directly related to triclosan use during the 40 years that it has been in use.20 The only link to resistance has been shown in vitro in Escherichia coli but in no other organism.20 Furthermore, it is thought that triclosan may affect another cellular target in addition to the enoyl reductase enzyme, thus, making targeted resistance mechanisms less likely.20 Long-term exposure to triclosan does not necessarily lead to drug resistance, perhaps giving triclosan an advantage over other agents used in biomaterial coating. To our knowledge there is no convincing evidence to date that the use of triclosan has led to the development of triclosan resistant or antibiotic resistant organisms. In this study 13 of 48 stents were malpositioned or they migrated into the enlarged rabbit prostatic fossa, a problem that also occurred in the original description by Fung et al.9 We found that the use of concomitant ultrasound was helpful to facilitate stent placement but it was not foolproof because some stents were still misplaced. The angled ureteral catheter aided in directing the guidewire into the bladder. None of the stent curls were voided out and previous study at our laboratory has demonstrated that 10 mm is the minimum diameter of an object that will be retained in the rabbit bladder.12

3.

4.

5.

6.

7.

8.

9.

10. 11.

12.

CONCLUSIONS Triclosan loaded ureteral stents decreased the number of UTIs as well as the total levels of urinary and device associated bacteria in a rabbit model of cystitis infected with P. mirabilis. These stents may prove clinically useful for the treatment of obstructive pyelonephritis in conjunction with parenteral antibiotics by providing drainage of the upper urinary tract in addition to local antibiosis. Triclosan loaded ureteral stents may prevent stent related UTIs and encrustation caused by Proteus. Clinical trials are currently under way. ADDENDUM The Triumph® stent is available for use in Canada but to date it is not available for sale in the United States.

13. 14.

15.

16.

17.

Abbreviations and Acronyms cfu ⫽ colony forming units hpf ⫽ high power field UTI ⫽ urinary tract infection REFERENCES 1.

2.

Tieszer, C., Reid, G. and Denstedt, J.: Conditioning film deposition on ureteral stents after implantation. J Urol, 160: 876, 1998 Kunin, C. M.: Urinary Tract Infections: Detection, Prevention and Management. Baltimore: Williams and Wilkins, 2004

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20.

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Morris, N. S., Stickler, D. J. and Winters, C.: Which indwelling urethral catheters resist encrustation by Proteus mirabilis biofilms? Br J Urol, 80: 58, 1997 Barbolt, T. A.: Chemistry and safety of triclosan, and its use as an antimicrobial coating on Coated VICRYL* Plus Antibacterial Suture (coated polyglactin 910 suture with triclosan). Surg Infect (Larchmt), suppl., 3: S45, 2002 Rothenburger, S., Spangler, D., Bhende, S. and Burkley, D.: In vitro antimicrobial evaluation of Coated VICRYL* Plus Antibacterial Suture (coated polyglactin 910 with triclosan) using zone of inhibition assays. Surg Infect (Larchmt), suppl., 3: S79, 2002 Storch, M., Perry, L. C., Davidson, J. M. and Ward, J. J.: A 28-day study of the effect of Coated VICRYL* Plus Antibacterial Suture (coated polyglactin 910 suture with triclosan) on wound healing in guinea pig linear incisional skin wounds. Surg Infect (Larchmt), suppl., 3: S89, 2002 Skaare, A. B., Kjaerheim, V., Barkvoll, P. and Rolla, G.: Does the nature of the solvent affect the anti-inflammatory capacity of triclosan? An experimental study. J Clin Periodontol, 24: 124, 1997 Stickler, D. J., Jones, G. L. and Russell, A. D.: Control of encrustation and blockage of Foley catheters. Lancet, 361: 1435, 2003 Fung, L. C., Mittelman, M. W., Thorner, P. S. and Khoury, A. E.: A novel rabbit model for the evaluation of biomaterial associated urinary tract infection. Can J Urol, 10: 2007, 2003 Bhargava, H. N. and Leonard, P. A.: Triclosan: applications and safety. Am J Infect Control, 24: 209, 1996 Matsuyama, T., Takagi, Y., Nakagawa, Y., Itoh, H., Wakita, J. and Matsushita, M.: Dynamic aspects of the structured cell population in a swarming colony of Proteus mirabilis. J Bacteriol, 182: 385, 2000 Watterson, J. D., Cadieux, P. A., Beiko, D. T., Cook, A. J., Burton, J. P., Harbottle, R. R. et al: Oxalate-degrading enzymes from Oxalobacter formigenes: a novel device coating to reduce urinary tract biomaterial-related encrustation. J Endourol, 17: 269, 2003 McMurry, L. M., Oethinger, M. and Levy, S. B.: Triclosan targets lipid synthesis. Nature, 394: 531, 1998 Mittelman, M., Bucay-Cuto, W., Li, J., Miller, K., Schuermann, J. and Denstedt, J. D.: In vitro antimicrobial release profile of a new triclosan-eluting ureteral stent. J Urol, 171: 443, 2004 Moss, T., Howes, D. and Williams, F. M.: Percutaneous penetration and dermal metabolism of triclosan (2,4, 4=-trichloro-2=-hydroxydiphenyl ether). Food Chem Toxicol, 38: 361, 2000 Braoudaki, M. and Hilton, A. C.: Low level of cross-resistance between triclosan and antibiotics in Escherichia coli K-12 and E. coli O55 compared to E. coli O157. FEMS Microbiol Lett, 235: 305, 2004 Chuanchuen, R., Beinlich, K., Hoang, T. T., Becher, A., Karkhoff-Schweizer, R. R. and Schweizer, H. P.: Cross-resistance between triclosan and antibiotics in Pseudomonas aeruginosa is mediated by multidrug efflux pumps: exposure of a susceptible mutant strain to triclosan selects nfxB mutants overexpressing MexCD-OprJ. Antimicrob Agents Chemother, 45: 428, 2001 Heath, R. J. and Rock, C. O.: A triclosan-resistant bacterial enzyme. Nature, 406: 145, 2000 McBain, A. J., Ledder, R. G., Sreenivasan, P. and Gilbert, P.: Selection for high-level resistance by chronic triclosan exposure is not universal. J Antimicrob Chemother, 53: 772, 2004 Russell, A. D.: Biocide use and antibiotic resistance: the relevance of laboratory findings to clinical and environmental situations. Lancet Infect Dis, 3: 794, 2003