Effectiveness of topical antibiotics on Staphylococcus aureus biofilm in vitro Martin Desrosiers, M.D., *§ Zohra Bendouah, M.Sc.* and Jean Barbeau, Ph.D.# (Canada)
ABSTRACT
Background: In vitro biofilm-producing capacity in isolates of Staphylococcus aureus and Pseudomonas aeruginosa collected from the sinus cavities after endoscopic sinus surgery (ESS) are associated with a poor outcome in patients with chronic rhinosinusitis (CRS). However, conventional oral antibiotic therapy is frequently ineffective in eradicating bacteria in the biofilm form. Increasing the concentration of antibiotics may offer a means of countering this resistance. The aim of this study was to determine the in vitro activity of moxifloxacin (MOXI) against S. aureus in biofilm form (recovered from patients with CSR at least 1 year post-ESS).
Method: This study was performed in a research microbiology laboratory, where five isolates of S. aureus with known biofilm-forming capacity were cultured in Tryptic Soy Broth 0.5% glucose in 96-well plates at 37°C for 24 hours. After visual confirmation of biofilm formation, plates were incubated in phosphate-buffered saline (PBS) or with MOXI at concentrations of 0.1× , 1×, 100×, and 1000× minimal inhibitory concentration (MIC) for an additional 24 hours. Biofilm from 3 wells of each concentration were collected and sonicated and the number of viable bacteria was determined by serial dilution and plating.
Results: After incubation, the number of viable bacteria was similar for nontreated and MOXI-treated biofilms at MIC and sub-MIC levels. However, MOXI at 1000 × (0.1-0.2 mg/mL) gave a 2 to 2.5 log reduction in number of viable bacteria.
Conclusion: In vitro results show that increased concentrations of antibiotics, easily attainable in topical solutions, are effective in killing bacteria in bacterial biofilms. This suggests a role for topical antibiotic therapies in the treatment of biofilm infections. (AM J Rhinol 21, 149-153, 2007; doi: 10.2500/ajr.2007.21.3007)
Chronic rhinosinusitis (CRS) is a chronic disease of the paranasal sinuses characterized by persistent inflammation with bacterial colonization and infection. Current therapeutic recommendations for the management of CRS rely initially on medical management with oral antibiotics and topical or oral corticosteroids. For individuals failing to responds to these therapies, endoscopic sinus surgery is recommended for removing diseased tissue and improving drainage.
Cultures of individuals with chronic rhinosinusitis not responding to surgery have shown higher than usual incidences of Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumonia, and Haemophilus influenzae.1,2 On sensitivity testing, these usually are susceptible to available oral agents. However, oral antibiotic therapy frequently is unsuccessful in clearing CRS, particularly after ESS. The failure of antibiotic therapy in CRS is somewhat surprising, given the high success rate of antibiotics in the treatment of acute bacterial maxillary rhinosinusitis.
Biofilms have implicated in CRS and may help explain a portion of these phenomena. The presence of bacterial biofilms on the mucosa of patients with CRS and its role in sinus disease now has been established using a variety of techniques. Using scanning and transmission electron microscopy, Palmer,3 Ferugson4, and Ramadan5 have shown the presence of biofilm on the sinus mucosa. Using confocal laser microscopy with fluoresence in situ hybridization technique for identification of bacterial species, Hunsaker et al.6 have shown the presence of H. influenzae, S. pneumoniae, and S. aureus. Our group has shown a correlation in vitro biofilm-producing capacity by S. aureus and unfavorable evolution after ESS, 7 suggesting that biofilm-producing capacity may play a role in the pathogenesis of CRS.
The presence of bacterial biofilms may help explain the lack of effectiveness of oral antibiotics. By virtue of their nature, bacteria in biofilms are much less susceptible to antibiotics. Minimally inhibitory concentrations (MIC) effective on an organism in planktonic form are ineffective against the same organism when present in the biofilm form. Increasing the concentration of antibiotics may offer a means of countering this resistance.8 Although high concentrations of antibiotics are difficult to attain in serum because of risks of toxicity, antibacterials can be delivered safely in high concentrations when applied topically. Given that because after sinus surgery, the mucosa of the sinus cavities is accessible to topical therapy, it seems reasonable to evaluate the effect of topical antibiotics on bacteria from sinus isolates in the biofilm form.
Although topical antibiotic therapy has been attempted previously as therapy, newer broad-spectrum agents may offer an alternative to agents such as topical tobramycin. Moxifloxacin (MOXI) is a new enantiomerically pure 8-methoxyquinolone that was first synthesized by Petersen and colleagues9 with a broad-spectrum of antibacterial activity against Gram-positive and with first-generation quinolones, it has higher potency and pharmacodynamic activity. 11,12
We wish to explore the efficacy of topical MOXI on mature bacterial biofilms of S. aureus using isolates obtained from individuals with CRS. The aim of this study was to assess the in vitro activity of MOXI against clinical isolates of S. aureus in biofilm form.
MATERIALS AND METHODS
This study was approved by the Ethical Review Board for human subjects of the Centre Hospitalier de l'Université de Montréal.
Organisms
Four isolates previously recovered from patients with bad outcome after ESS for CRS with or without sinonasal polyposis and an isolate of S. aureus (LSPQ2520) obtained from the provincial reference laboratory were used. These isolates have been used in our previous studies of biofilm-forming capacity of sinus isolates and were selected again for biofilm-forming capacity. This population and collection method has been described previously. 13
Antimicrobial Agent
MOXI was supplies as a dry powder for laboratory use by Bayer Canada.
MIC Determination
MIC to MOXI was determined for each of the organisms studied. MICs are defined as the lowest concentration of an antimicrobial that will inhibit the invisible growth of a micro-organism after overnight incubation. MICs were determined by a standard macrodilution method.14 In previous published studies of acute bacterial sinusitis, MIC90 of MOXI for isolates of S. aureus was 0.12 μg/mL. 15,16
Biofilm Formation
Mature biofilm formation was performed on a 96-well culture plaque in Tryptic Soy Broth 0.5% glucose at 37°C for 24 hours as previously described.13 After visual confirmation of biofilm formation, the wells were washed three times with sterile phosphate-buffered saline (PBS) under aseptic conditions to eliminate nonadherent bacteria.17 Plates were then incubates for an additional 24 hours at 37°C in either sterile PBS or with PBS/MOXI solution at concentrations of 0.1×, 1×, 100×, and 1000× MIC. Wells with sterile PBS free of MOXI were used as controls. After incubation, wells were washed again three times with PBS. The biofilm then was collected to determine viable bacteria. Biofilms from 3 wells of each concentration were collected by scraping the well with a swab, and placed in 1 mL of PBS. The swab was sonicated for 2 minutes to free bacteria, and the biofilm form the 3 wells was pooled together. The number of viable bacteria was determined by serial dilution and plating. All experiments were performed in triplicate.
Analysis
Average colony-forming units (CFU) for the three samples tested were expressed as log of CFU per milliliter. Variability is expressed as standard error of the mean (SEM).
RESULTS
The susceptibilities (MIC) to MOXI of the bacterial strains tested are shown in Table 1. These range from 0.0937 to 0.1875 μg/mL. These MIC levels are comparable with levels previously reported for isolates of S. aureus recovered in trial of acute bacterial maxillary sinusitis. Results for each species are expressed in graphical form in Fig. 1. For all strains tested, MOXI at sub-MIC and MIC levels had no effect on established biofilm. At concentrations above MIC, there appeared to be a dose-related reduction in number of CFUs. Maximal effect was noted with MOXI at 1000× (100-200 μg/mL); which gave a 2-2.5 log reduction in number of viable bacteria within the biofilm. Despite this reduction, MOXI was incapable of eradicating completely viable bacteria in the mature biofilms.
DISCUSSION
We have performed an in vitro assessment of the effectiveness of differing concentrations of topical MOXI on killing S. aureus contained in a bacterial biofilm. Sub-MIC and MIC levels of MOXI have no effect on S. aureus contained in mature biofilms, confirming previous reports.17 However, our results do suggest that above-MIC concentrations of MOXI have some effectiveness in reducing viable bacteria in mature biofilms of S. aureus, suggesting that antibiotic therapy with supra-MIC level of MOXI and other antibiotics actually may have a role in the management of chronic sinusitis. However, delivery of supra-MIC levels of MOXI to the sinus cavities via an oral route will be difficult. After administration of a standard 400-mg dose, concentration of MOXI in the sinus mucosa , even though approximately double that of serum levels, do not exceed 10μg/mL at peak. For the isolates in our study, this would be ~50-100× MIC.18
Delivery via a topical route may represent an option to oral administration, particularly after ESS. After ESS, the sinus passages freely communicate with the nasal passages, allowing solutions applied within the nose to penetrate the sinus cavities (Fig. 2). Topical administration offers a potential for deposition of medication at higher concentrations directly on the surface of the biofilm.
Topical therapies previously have been recommended for post-ESS patients but have met with varying rates of success. A sole perspective, placebo-controlled trial of nebulized tobramycin for the treatment of chronic sinusitis refractory to medical and surgical therapy did not show any benefit of tobramycin over placebo,19 although a retrospective case series suggested effectiveness in certain cases.20 Criticisms to these trials include differences in patients populations treated (stable chronic refractory patients vs. acute exacerbations), the use of an agent (tobramycin) with limited efficacy against Gram-positive agents such as S. aureus, and, mainly, the absence of a precise measure of the quantity and concentration of antibiotic delivered to the sinus cavity via the apparatus used for topical administration.
It is noteworthy that MOXI at these concentrations did not completely eradicate bacteria in biofilms. Additional works to evaluate the postantibiotic effect on the biofilm remains to be determined. Nevertheless, our results suggest that supra-MIC levels of antibiotics represent an interesting area of study as we seek alternate means of improving the management of sever CRS.
CONCLUSION
In vitro results show that increased concentrations of antibiotics, easily attainable in topical solutions, are effective in killing bacteria in mature bacterial biofilms. This suggests a potential role for topical antibiotic therapies in the management of biofilm injections and that additional exploration of this area is warranted.
ACKNOWLEDGMENTS
We thank the Foundation Antoine Turmel for their generous support of our research activities.
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Desrosiers M, and Salas-Prato M. Treatment of chronic rhinosinusitis refractory to other treatments with topical antibiotic therapy delivered by means of a large-particle nebulizer. Results of a controlled trial. Otolaryngol Head Neck Surg 125:265-269, 2001.
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Acute bacterial sinusitis is an infection of the sinus cavities caused by bacteria.
It usually is preceded by a cold, allergy attack, or irritation by environmental pollutants.
Unlike a cold, or allergy, bacterial sinusitis requires a
physician's diagnosis and treatment with an antibiotic to cure the
infection and prevent future complication.
Acute bacterial sinusitis is an infection of the sinus cavities caused by bacteria.