The University of Texas M. D. Anderson Cancer Center, Department of Infectious Diseases, Infection Control and Employee Health, 1515 Holcombe Blvd, Houston, TX 77030, USA
Received 2 July 2004; returned 4 August 2004; revised 28 September 2004; accepted 6 October 2004
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Abstract |
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Methods: We determined the antimicrobial efficacy and cytotoxicity of ETT and UC segments coated, through an instant dip method, with gendine. Using the modified KirbyBauer method, gendine-coated devices showed zones of inhibition of 15 mm against methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Escherichia coli and Candida parapsilosis.
Results: Gendine-coated endotracheal tubes (GND-ETT) soaked in bronchoalveolar fluid (BAL) and incubated at 37°C maintained a zone of inhibition of 15 mm against MRSA and P. aeruginosa for at least 3 weeks. Similarly, gendine-coated urinary catheters (GND-UC), soaked in urine, maintained a zone of inhibition of
15 mm against E. coli for 8 weeks. Using the minimum essential media elution method in mouse fibroblast cells, GND-ETT and GND-UC were found to be non-cytotoxic. Gendine-coated UC significantly reduced the amount of viable MRSA, E. coli or C. parapsilosis organisms adhering to their surfaces when compared with silver/hydrogel-coated urinary catheters or control uncoated catheters (P < 0.01). Similarly GND-ETT significantly reduced the adherence of the same organisms as well as P. aeruginosa when compared with control (P
0.02).
Conclusions: GND-ETT and GND-UC impregnated using an instantaneous dip method, were shown to have broad-spectrum activity, prolonged antimicrobial durability and high efficacy in inhibiting adherence of organisms commonly causing nosocomial pneumonia and urinary tract infection. Furthermore, these coated devices were shown to be non-cytotoxic.
Keywords: Gentian Violet , medical devices , novel antiseptics , antimicrobial activity , bacterial adherence
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Introduction |
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Similarly, studies have shown that mechanical ventilation is a major contributor to nosocomial pneumonia, and that the infection is specifically related to the use of endotracheal tubes.4,5 Ventilator-associated pneumonia is a serious complication and is associated with a mortality rate up to 48%.5
Invasive medical devices increase the risk of infection because they bypass normal host defences and allow microorganisms to access normally sterile environments.6 The surfaces of these devices, external or intraluminal, act as a substrate that allows for the adherence of these microorganisms.7 After the initial layer of organisms adheres to the surface, they begin to form a biofilm. Biofilm-forming cells adhere to each other and become a viable cluster by embedding themselves in a polysaccharide matrix, which protects them from the host's defences and antimicrobial therapy.5,6,8 Problems arising from these infections complicate the course of treatment and increase the length of patient stay in the hospital and overall cost.
In order to prevent colonization of urinary catheters and endotracheal tubes, we coated these devices with a novel antiseptic agent, referred to as gendine. This agent consists of a combination of Gentian Violet and chlorhexidine dissolved in an impregnating solvent. Gendine was used to impregnate the endotracheal tubes and urinary catheters through a rapid, instant-dipping method. The purpose of this study was to test gendine-impregnated devices in terms of their cytotoxicity and antimicrobial efficacy against a wide spectrum of infectious organisms.
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Materials and methods |
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Gendine is prepared by a proprietary method described in a patent application, which is currently pending.9 One-centimetre segments of endotracheal tubes and urinary catheters were instantaneously dipped into the gendine solution, so that both internal and external surfaces were coated. The pieces were left to dry overnight, at room temperature, on a sterile surface under a fume hood. The pieces were then washed using mild detergent and deionized water to remove any possible loosely attached antiseptic off the surface of the coated segments. The pieces were then left to dry for 4 h under a fume hood at room temperature.
Baseline zones of inhibition
Using a modified KirbyBauer method,10 baseline antimicrobial activity was assessed by measuring the zones of inhibition created by duplicates of impregnated segments that were vertically embedded in MuellerHinton agar plates coated with one of the following organisms: MRSA, Candida albicans, Candida parapsilosis, vancomycin-resistant enterococci (VRE), Pseudomonas aeruginosa and Escherichia coli. The zones of inhibition were measured and recorded as the diameter (mm) across the centre of the embedded segments.
Antimicrobial durability
The antimicrobial efficacy of gendine-coated endotracheal tubes (GND-ETT) and gendine-coated urinary catheter (GND-UC) segments was assessed over time. The segments were placed in sterile 50 mL polystyrene tubes (Falcon), containing 10 mL sterile bronchoalveolar lavage (BAL) or urine, respectively, and were incubated at 37°C. A 10 mL volume was used to ensure the complete immersion of all of the pieces contained in the tube. Segments were tested in duplicate at weekly intervals, as the soaking fluids were replaced with fresh fluids. Zones of inhibition were determined using the modified KirbyBauer method against the same organisms mentioned above.
Adherence testing
We evaluated bacterial adherence to the surface of gendine-coated and control uncoated segments, as well as that of silver hydrogel UC. Six segments of each device were tested per organism. We used a modification of a previously published method for testing adherence and biofilm formation on silicone discs.8 The sterile device segments were placed into sterile 5 mL snap-top tubes containing 1 mL of plasma. The tubes were then placed into the incubator for 24 h at 37°C. The plasma was then removed from the tubes, leaving the pieces inside the tubes, and was replaced with 1 mL of MuellerHinton Broth (MHB), that was inoculated with bacteria. The inoculum was prepared as follows: five colonies of freshly subbed bacteria were placed in 50 mL of MHB (turbidity equivalent to that of a 0.5 McFarland Standard; approximately 1.3 x 108 cfu/mL), and used immediately. The tubes were then placed in the incubator for 24 h at 37°C. The MHB was then removed and replaced with 1 mL of 0.9% saline solution and the tube was shaken on a rocker, in the 37°C incubator for 30 min as a washing step. The catheter segments were then removed from the washing saline, and placed into sterile 15 mL tubes containing 5 mL of 0.9% sterile saline solution and sonicated for 15 min. After sonication, the tubes containing the catheters were vortexed for 60 s. A 100 µL volume of the sample was pipetted and spread onto a trypticase soy agar plate with 5% sheep blood (this was the 1:50 dilution). The plates were placed in the incubator for 24 h, and then the colonies were counted. A value of 100 cfu was used for any plate that had at least 100 counted colonies.
Cytotoxicity testing
Cytotoxicity tests were carried out (BEC Laboratories, Inc., Toledo, OH, USA) using the United States Pharmacopoeia (USP) standard minimum essential media (MEM) elution test against L929 mouse fibroblast cells. Using the polymeric preparation for the endotracheal tubes, a ratio of 4.0 g gendine-coated endotracheal tubes per 20 mL of 5% bovine serum solution in MEM was used to extract the sample. The elastomer preparation ratio was used for the urinary catheters. Two grams of GND-UC/20 mL 5% bovine serum solution in MEM was used to extract the sample. The samples were extracted for 24 h at 37°C. Duplicate 5 mL volumes of the sample MEM extract were added to separate monolayers of L929 mouse fibroblast cells. The cells were incubated at 37°C for 48 h. The cells were observed for cytotoxic effects. For the negative control, 20 mL of a 5% bovine serum solution in MEM was used to extract 60 cm2 high density polyethylene (USP) for 24 h at 37°C. For the positive control, 20 mL of a 5% bovine serum solution in MEM was used to extract 120 cm2 latex for 24 h at 37°C. Cytotoxicity was determined based on a scoring system between zero and four, whereby scores between zero and two indicated no cytotoxicity and scores of three and four indicated cytotoxicity.
Statistical analysis
All statistical analyses were carried out using the statistical computing package SPSS (version 11.0 for Windows; SPSS Inc., Chicago, IL, USA).
Continuous variables pertaining to the number of colony-forming units representing adherence of organisms to the surface of catheter segments were compared with Student's t-test or MannWhitney test.
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Results |
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Table 1 shows the results of the baseline tests for the gendine-coated pieces against various organisms. The numbers represent a measurement of the diameter of the zone of inhibition, in millimetres, including the diameter of the pieces embedded in the agar (the urinary catheter segments were 8 mm in diameter and the endotracheal tube segments 14 mm in diameter). Zones of inhibition produced by GND-UC segments ranged between 17 and 24 mm in diameter, and those produced by GND-ETT were 2335 mm.
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Table 2 shows the results of the cytotoxicity tests for GND-ETT and GND-UC. Based on the scoring method outlined for elution tests, the tested segments scored grade 1 on the 04 scale, indicating no cytotoxicity. The positive controls for both tests scored grade 3. The negative controls scored grade 0.
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Figure 1 shows that the durability of the GND-UC lasted for at least 8 weeks, with zones 15 mm for 4 weeks. Zones of inhibition for the three organisms tested, C. albicans, VRE and E. coli, measured 1823 mm at baseline. After being immersed in urine which was changed at weekly intervals, GND-UC still produced zones of inhibition that measured 1117 mm at 8 weeks. C. albicans consistently maintained larger zones of inhibition than VRE, which consistently maintained larger zones of inhibition than E. coli (Table 1). C. albicans, at baseline, had an average zone of inhibition of 23 mm, and at 8 weeks ended with an average of 17 mm. Vancomycin-resistant enterococci, at baseline, had an average zone of inhibition of 20 mm, and at 8 weeks maintained an average of 12 mm. E. coli, at baseline, had an average zone of inhibition of 18 mm, and reached an average of 11 mm after 8 weeks.
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Figure 2 shows the durability study for endotracheal tubes. The four organisms tested for durability, MRSA, C. parapsilosis, P. aeruginosa and E. coli, had average zones of inhibition, at baseline, that ranged from 23 to 32 mm in diameter. All of the organisms tested maintained zones 15 mm after 3 weeks of immersion in BAL, which was changed at weekly intervals. After 3 weeks of immersion in BAL, MRSA, C. parapsilosis, P. aeruginosa and E. coli maintained average zones of inhibition of 20, 19, 15 and 19 mm in diameter, respectively.
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Figure 3 shows a comparison of bacterial adherence to GND-UC catheters, silver/hydrogel-coated urinary catheters, and control uncoated silicone urinary catheters. The GND-UC significantly reduced the amount of viable organisms adhering to their external and internal surfaces, except for P. aeruginosa, when compared to the uncoated control and silver/hydrogel-coated catheters (P < 0.01). The silver/hydrogel-coated segments were similar to the controls for both MRSA and C. parapsilosis. There was some non-significant reduction in the number of adherent E. coli on the silver/hydrogel catheter surface, whereby the average colony count was 4167 for the silver/hydrogel catheters, and >5000 for the controls.
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Figure 4 compares GND-ETT to uncoated control segments. In comparison to the control segments, the coated segments achieved complete inhibition of adherence of MRSA, C. parapsilosis and E. coli (P=0.03). The only organism that adhered to the coated tubes was P. aeruginosa, however its adherence was significantly reduced compared with the uncoated control segments (P=0.02). All of the organisms tested had confluent growth with biofilm formation around control segments with an average growth of > 5000 cfu per segment.
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Discussion |
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Therefore, the properties of gendine allow for a rapid and simple method of application to the surfaces of endotracheal tubes and urinary catheters after the manufacturing of the device has been completed. Furthermore, after the device is instantly dipped, gendine does not appear to weaken or distort the physical characteristics of the devices.
This antiseptic can be used to coat medical devices made of different polymers. In this and other studies testing gendine, devices made of polyvinylchloride, silicone and various polyurethanes have been successfully coated.9 These polymers make up the majority of medical implant devices including endotracheal tubes, urinary catheters, central-venous catheters, biliary stents and chest tubes. As a result, gendine may be used to confer antimicrobial properties to a wide variety of invasive devices.
Despite sterile precautions and the use of a variety of interventions, device-related infections continue to occur. Studies have shown that mechanical ventilation is a major contributor to nosocomial pneumonia, and that the infection is specifically related to the use of endotracheal tubes.4
As a result of endotracheal tube use, 1540% of patients who are intubated for >48 h develop ventilator-associated pneumonia, with rates reaching as high as 65% in the ICU.5,6
Ventilator-associated pneumonia is a serious problem to manage, with a mortality rate up to 48%.5
Craig & Connelly studied a large group of ICU patients and matched those with pneumonia with those who did not develop pulmonary infection.11
Patients with pneumonia stayed in the ICU three times longer and had a four-fold greater mortality (P < 0.001). Endotracheal tubes act as a bridge connecting the oropharyngeal environment with the sterile bronchoalveolar space. Organisms migrate from the oropharyngeal space to the bronchoalveolar environment by either micro-aspiration, where secretions accumulate around the cuff of the endotracheal tube,12
or by attaching themselves on the endotracheal tubes and migrating along this polymer.4,13
Coating endotracheal tubes with an effective antimicrobial agent, which also coats the cuff, is likely to decrease the risk of infection. The most common organisms associated with ventilator-associated pneumonia in intubated patients are MRSA and P. aeruginosa, followed by other enteric organisms, such as E. coli.14
As shown in our study, GND-ETT segments were highly efficacious against MRSA, P. aeruginosa and E. coli with prolonged antimicrobial efficacy, even after being immersed in bronchoalveolar lavage. The in vitro large zones of inhibition (15 mm) created by devices coated with antimicrobial agents have been shown to be predictive of their clinical efficacy.15
In our model, the endotracheal tubes coated with gendine were found to have antimicrobial durability extending to at least 3 weeks with zones of inhibition of >15 mm against MRSA, P. aeruginosa, E. coli and C. parapsilosis. Because endotracheal tubes do not remain in place longer than 3 weeks in intubated patients, the coating of endotracheal tubes with an effective antiseptic agent, such as gendine, could be an important intervention that could be added to other beneficial interventions, such as continuous aspiration of subglottic secretions, to prevent nosocomial pneumonia.16
Urinary tract infections (UTI) are the most common nosocomial infections occurring in critically ill patients and at least 95% of those are urinary catheter-associated.17 It is estimated that 9 000 000 episodes of nosocomial UTI occur annually in the United States, increasing the cost of health care by more than $500 000 000 annually.18 The leading organisms causing nosocomial UTI are enteric organisms such as E. coli followed by enterococci, including VRE, and C. albicans.19 The only approved anti-infective urinary catheter in the United States is the silver alloy urinary catheter which has been shown to decrease the relative risk of UTI by 32% (from 3.1/100 catheter days to 2.13/100 catheter days, P < 0.01) with a cost savings of $14 456 per 100 catheters.1,20,21 Our data show that the silver alloy catheters are able to decrease the adherence of E. coli by around 30% when compared with control. However, the gendine-coated catheters were able to decrease the adherence of E. coli and C. parapsilosis by at least 90% and the adherence of MRSA by at least 60% (P < 0.05). Given the lack of cytotoxicity of the gendine-coated urinary catheters and their prolonged antimicrobial durability, which can extend to at least 8 weeks in urine, these coated catheters could have an impact on preventing nosocomial UTI in hospitalized patients as well as spinal cord injury patients who require prolonged urinary catheterization.
While concerns of developing antibiotic resistance have been raised for catheters coated with antibiotics, gendine is an antiseptic made of a combination not used therapeutically in the treatment of systemic infections, and should not contribute to the risk of developing antibiotic-resistant bacteria. Although catheters coated with antibiotics have been shown to be effective in preventing colonization and infections associated with vascular and urinary catheters,2224 concerns related to the emergence of antibiotic-resistant organisms may limit their clinical use.25 In an in vitro study evaluating antibiotic resistance associated with antibiotic coated catheters, Tambe et al. suggested that chlorhexidine does not significantly change bacterial resistance.25 Furthermore, antibiotics such as minocycline and rifampicin are highly active against staphylococcal skin organisms associated with vascular catheter infections but are less active against Gram-negative organisms, often associated with nosocomial endotracheal tube-related pneumonias and urinary catheter associated infections. Both Gentian Violet and chlorhexidine possess antimicrobial activity against a broad range of bacteria and yeasts.
The in vitro cytotoxicity tests carried out on the coated endotracheal tubes and urinary catheters indicated that the technology has the potential to be safe. Further evidence suggesting that gendine may be safe stems from the variety of medical applications using the component antiseptics chlorhexidine or Gentian Violet without signs of toxicity. Gentian Violet, in addition to being used for cell staining procedures, has been used topically to treat skin lesions, to coat the oral cavity of neonates who developed thrush, and also as an oral rinse for patients with oropharyngeal candidiasis.26,27 Chlorhexidine is used for an even wider variety of medical applications. Chlorhexidinealcohol solutions are used to disinfect skin surgical sites, and are used as hand scrubs. A chlorhexidine-based oral rinse is used for the treatment of tooth decay and periodontal infections. Chlorhexidine has also been successfully incorporated into central venous catheters, as with the chlorhexidine gluconate and silver sulfadiazine coated vascular catheter which has been shown to be highly effective in decreasing catheter-related infections.28,29 However, chlorhexidine-silver sulfadiazine coated central venous catheters have been banned from the Japanese market due to their association with anaphylactic shock. In addition, further cytotoxicity testing is needed either through in vivo testing or local cytotoxicity testing that allows intimate contact of cells with the treated medical devices.
In conclusion, gendine's ability to coat various polymers and devices instantaneously, its non-cytotoxicity, broad-spectrum anti-adherence activity, and long-term efficacy make it a unique antiseptic that is highly effective in coating medical devices. The antimicrobial activity of gendine, applied to coating devices, decreases the risk of device colonization, which may in turn decrease the rates of nosocomial infections and their associated morbidity and mortality, resulting ultimately in a cost benefit. Future in vivo and clinical trials are needed to determine whether gendine will be able to reduce the rates of infection and colonization associated with the use of urinary catheters and endotracheal tubes, resulting in increased benefits to patients.
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Footnotes |
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References |
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