Introduction
Tympanostomy tube insertion is the most commonly performed surgical procedure in children younger than 15 years. Purulent otorrhoea is the most common complication following tympanostomy tube insertion, occurring in 9 to 34 per cent of patients.Reference Barfoed and Rosborg1–Reference Younis, Lazar and Long5 The causative pathogens in young children with acute otorrhoea are the same as those found in paediatric patients with acute otitis media and an intact tympanic membrane.Reference Bluestone and Klein6
There has been a steady increase in the number of cases of otorrhoea caused by methicillin-resistant Staphylococcus aureus (MRSA).Reference Jang, Song and Wang7 Recently, concern has been expressed over the increasing incidence of community-acquired MRSA infection presenting in paediatric patients with otitis media with otorrhoea.Reference AI-Shawwa and Wegner8 An increased incidence of MRSA tympanostomy tube otorrhoea has also been observed.Reference Coticchia and Dohar9 Bacterial biofilm formation has been implicated in the high rate of persistent otorrhoea after tympanostomy tube insertion.Reference Karlan, Mufson, Grizzard, Buscemi, Hench and Goldberg10–Reference Johnson, Roberts, Olsen, Moyer and Stamin12 Once a staphylococcal biofilm has formed on an implanted medical device or damaged tissue, it is difficult to disrupt. Biofilm-infected implants must often be removed and replaced, placing the patient at increased risk of complications.Reference Raad, Darouiche, Hachem, Abi-Said, Safar and Darnule13 Current antimicrobial therapies for biofilms have proven largely unsuccessful.Reference Donlan and Costerton14
An ideal tympanostomy tube material would be well tolerated by the patient and resistant to bacterial adhesion. However, it has been suggested that currently used tube materials may be influential in the development of otorrhoea. Silicone is currently the most commonly used material for tympanostomy tubes. The use of fluoroplastic tympanostomy tubes has resulted in a significant reduction in otorrhoea incidence, compared with silicone tympanostomy tubes.Reference Karlan, Mufson, Grizzard, Buscemi, Hench and Goldberg10 Silver ions inhibit the growth of staphylococcus, streptococcus, Escherichia coli and pseudomonas, and the incorporation of silver oxide into tympanostomy tubes appears to decrease the long-term incidence of otorrhoea.Reference Chole and Hubbell15 The use of silver oxide confers to tympanostomy tubes the ability to prevent adherence and colonisation of selected bacteria, rather than affecting an already established infection.
The influence of silver oxide on MRSA is ill-defined. There is reason to believe that silver oxide may exert antibacterial effects against MRSA, since antibiotic coatings are effective against biofilm formation. For example, we have observed that tympanostomy tubes coated with a mixture of chitosan, piperacillin and tazobactam display effective resistance to the formation of ciprofloxacin-resistant pseudomonas biofilms.Reference Jang, Park, Cho, Choi and Park16
Vancomycin is not an aminoglycoside antibiotic but is often mistaken for one due to the ‘mycin’ suffix. In experimental animal models using large single vancomycin doses, there was no convincing evidence of ototoxicity from vancomycin.Reference Brummett17 In fact, one report of a patient who inadvertently received six 185-mg doses of vancomycin described no resulting auditory damage.Reference Mellor, Kingdom, Cafferkey and Keane18 Topical vancomycin has been found to be effective in patients with MRSA otorrhoea refractory to conventional antibiotic treatment; in addition, none of these patients showed any adverse effects during topical vancomycin treatment, and there were no statistically significant differences in mean bone conduction hearing thresholds as a result of treatment.Reference Jang, Song and Wang7
In the current study, we evaluated the efficacy of vancomycin-coated tympanostomy tubes in preventing MRSA biofilm formation in vitro.
Materials and methods
Vancomycin coating
To create the vancomycin coating, we used 90–95 per cent deacetylated, water-soluble chitosan (molecular weight <10 kDa; Ecobio, Gwangju, South Korea). Chitosan solutions of 5 per cent by weight and pH 4.5 were prepared by dissolution of chitosan in 0.2 M acetic acid. The mixture was stirred at 50°C for 2 hours to obtain a homogeneous polymer solution. The reaction mixture was then filtered through a fine cloth to remove air bubbles trapped in the viscous liquid. Vancomycin powder and a Paparella type one tympanostomy tube with an inner diameter of 1.14 mm (Medtronic Xomed, Jacksonville, Florida, USA) were added to the prepared chitosan solution to create a chitosan and vancomycin coated tympanostomy tube. The tube was rapidly transferred to a −20°C freezer for 8 hours to solidify the solvent and induce solid–liquid phase separation. The tympanostomy tubes were then transferred to a freeze-dryer and were lyophilised for five days to completely remove the solvent.
In vitro testing
We evaluated three sets each of vancomycin-coated, commercial silicone tubes (n = 5; Medtronic Xomed), commercial silver oxide coated silicone tubes with an inner diameter of 1.14 mm (n = 5; Activent Silic, Medtronic Xomed), and Paparella type one uncoated tympanostomy tubes with an inner diameter of 1.14 mm (as controls; n = 5; Medtronic Xomed).
Twenty clinical MRSA isolates were obtained from patients with otorrhoea due to chronic suppurative otitis media treated at the Chonnam National University Hospital, Gwangju City, South Korea, from March 2006 to February 2007.
Each isolate was grown to logarithmic phase in tryptone soy broth at 37°C for 24 hours, harvested by centrifugation, resuspended in a volume of tryptone soy broth and monitored spectrophotometrically to yield approximately 109 colony-forming units per millilitre. Before each experiment, several colonies were used to inoculate 5 ml tryptone soy broth at 37°C for 24 hours in ambient air. Subsequently, 100 µl of this culture was used to inoculate 50 ml of medium, from which biofilms were grown on tympanostomy tubes introduced by suspension from a stainless steel hook. After 72 hours' incubation at 37°C with continuous shaking at 60 rpm, the inoculated tryptone soy broth was refreshed with sterile tryptone soy broth.
After a total incubation period of six days, the tympanostomy tubes were collected, dipped once in sterile 0.9 per cent NaCl and then gently dried by a minimal touch with a soft tissue, to wash off planktonic micro-organisms as well as any remaining tryptone soy broth. All tympanostomy tubes were immersed in fresh 2 per cent glutaraldehyde overnight. The tubes were then cut longitudinally into two segments for scanning electron microscopy.
The tubes were prepared for microscopy by critical point drying and gold sputter coating. All prepared specimens were investigated, for 30 minutes each, for MRSA biofilm formation on the surface of the tube.
Results
The surface of the uncoated control tympanostomy tubes showed diffuse MRSA biofilm formation on both the outer and inner surfaces (Figure 1).
Fig. 1 Scanning electron micrograph of the surface of an untreated silicone tympanostomy tube. Diffuse methicillin-resistant Staphylococcus aureus biofilm formation is evident at (a) lower and (b) higher magnification.
The silver oxide coated tubes showed no resistance to MRSA; their surfaces showed thick biofilms with crusts (Figure 2).
Fig. 2 Scanning electron micrograph of the surface of a silver oxide coated silicone tympanostomy tube. Methicillin-resistant Staphylococcus aureus biofilm formation is evident at (a) lower and (b) higher magnification.
In contrast, the surface of the vancomycin-coated tympanostomy tubes was devoid of biofilm (Figure 3).
Fig. 3 Scanning electron micrograph of the surface of a vancomycin-coated silicone tympanostomy tube. Almost complete absence of methicillin-resistant Staphylococcus aureus biofilm is evident at (a) lower and (b) higher magnification.
All of the tubes in each group showed similar findings.
Discussion
Methicillin-resistant S aureus otorrhoea is a growing concern. Intravenously administered vancomycin hydrochloride is the drug of choice in treating MRSA-infected patients. As an alternative approach, we evaluated the resistance of vancomycin-coated tympanostomy tubes to MRSA biofilm formation in vitro. Tubes coated with vancomycin were less prone to MRSA biofilm formation, compared with silver oxide coated tympanostomy tubes. Vancomycin was applied along with chitosan, a linear polysaccharide derived from crustacean shells and fungi cell walls, which has many reported medical applications including use as an implant coating.Reference Jang, Park, Cho, Choi and Park16, Reference Aimin, Chunlin, Juliang, Tinyin and Zhichao19–Reference Bumgardner, Wiser, Gerard, Bergin, Chesnutt and Marin21 The advantages of chitosan include biocompatibility, drug delivery and reported bacteriostatic properties.Reference Campbell, Song, Li, Nelson, Bottoni and Brooks22, Reference Hamilton, Yuan, Rigney, Chesnutt, Puckett and Ong23
Reduction in biofilm contamination could substantially reduce the incidence of otorrhoea. Chole and Hubbell have reported a decreased incidence of otorrhoea with the use of silver oxide coated silicone tympanostomy tubes, compared with plain silicone tympanostomy tubes.Reference Chole and Hubbell15 However, silver oxide coated tubes are also susceptible to biofilm formation.Reference Hamilton, Yuan, Rigney, Chesnutt, Puckett and Ong23, Reference Biedlingmaier, Samaranayake and Whelan24 In the present study, MRSA biofilm formation occurred on silver-treated tympanostomy tubes, indicating resistance of MRSA to silver oxide. Ion-bombarded silicone tympanostomy tubes and phosphorylcholine-coated tubes have been reported to resist biofilm formation.Reference Saidi, Biedlingmaier and Whelan25, Reference Licameli, Johnston, Luz, Daley and Kenna26 However, to date, these types of tubes have not been reported to resist MRSA biofilms.
• Bacterial biofilm formation has been implicated in the high rate of persistent discharge occurring after tympanostomy tube insertion
• The tympanostomy tube material may be an important factor in the development of aural discharge
• This in vitro study found that vancomycin-coated tympanostomy tubes resisted methicillin-resistant Staphylococcus aureus biofilm formation
• Further studies are required to establish the possible clinical value of this finding in human patients
Cağavi et al. studied microbial colonisation of vancomycin-coated catheters and silicone elastomer catheters.Reference Cağavi, Akalan, Celik, Gür and Güçiz27 Their results showed us that a coated catheter more important than the topical application of vancomycin to the shunt catheters (Regular vancomycin coated catheters are superior to silicone catheters that were topically immersed using vancomycin. They prevent bacterial colonization in some respect.)
Conclusion
This study compared vancomycin-coated tympanostomy tubes with silver oxide coated tubes and uncoated, control tubes as regards resistance to MRSA biofilm formation in vitro. The vancomycin coating inhibited MRSA biofilm formation. Further studies are necessary to investigate this potential in vivo.
Acknowledgement
This work was supported by a National Research Foundation grant funded by the Korean government Ministry of Education, Science and Technology (MEST) through the Research Center for Resistant Cells (R13-2003-009).
