Corticosteroids Directly Reduce Staphylococcus aureus Biofilm Growth: An In Vitro Study

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1 The Laryngoscope VC 2013 The American Laryngological, Rhinological and Otological Society, Inc. Corticosteroids Directly Reduce Staphylococcus aureus Biofilm Growth: An In Vitro Study Rachel Goggin, BMedSci(Hons); Camille Jardeleza, MD; Peter-John Wormald, MD; Sarah Vreugde, MD, PhD Objectives/Hypothesis: Clinical improvement in patients with chronic rhinosinusitis (CRS) treated with steroids alone has previously been ascribed to the steroids anti-inflammatory properties rather than any direct effect on the bacteria. The aim of this study was to determine if commonly used intranasal steroids directly reduce bacterial biofilm production in vitro. Study Design: In vitro comparative controlled trial. Methods: Staphylococcus aureus biofilms were grown on minimum biofilm eradication concentration device pegs and treated with the commonly prescribed CRS topical steroids fluticasone, mometasone, or budesonide. These were dissolved in vehicle solvents and added to cerebrospinal fluid (CSF) broth. Concentrations (including therapeutic doses) tested for fluticasone and mometasone ranged from 25 lg/200 ll to 400 lg/200 ll, and from 16 lg/200 ll to 2000 lg/200 ll for budesonide. Control pegs were exposed to equivalent volumes of the appropriate solvent/csf broth. Confocal scanning laser microscopy and COMSTAT software were used to quantify biofilms at 24 hours after treatment. Results: Significant differences from control were found for fluticasone at 400 lg/200 ll (difference lm 3 /lm 2, P 5.007), mometasone at 300 lg/200 ll and 400 lg/200 ll (difference lm 3 /lm 2, P 5.006, and difference lm 3 /lm 2, P 5.034, respectively), and budesonide at 750 lg/200 ll, 1000 lg/200 ll and 2000 lg/200 ll (difference lm 3 /lm 2, P 5.038, difference , P 5.009, and difference lm 3 /lm 2, P 5.029, respectively). Conclusions: The concentrations of 400 lg/200 ll of fluticasone, 300 lg and 400 lg/200 ll of mometasone, and 750 lg, 1,000lg, and 2,000 lg/200 ll of budesonide directly reduce biofilm production in vitro, outside of the inflammatory milieu. Key Words: Chronic rhinosinusitis, biofilms, Staphylococcus aureus, steroids, fluticasone, mometasone, budesonide. Level of Evidence: NA Laryngoscope, 124: , 2014 INTRODUCTION The current accepted treatment for chronic rhinosinusitis (CRS) is medical, and if this fails, surgical. However, there is a small percentage of patients in whom CRS tends to recur despite maximal medical therapy and surgery. 1 CRS is the fifth most common diagnosis generating an antibiotic prescription, 2 adding significantly to medical costs and to the increasing problem of antibiotic resistance. 3,4 Staphylococcus aureus, which is present in up to 50% of CRS patients, 5,6 contributes to the persistence and recalcitrance of CRS 7 9 and is especially difficult to eradi- From the Department of Surgery Otorhinolaryngology, Head and Neck Surgery, Queen Elizabeth Hospital and the University of Adelaide, Adelaide, South Australia, Australia. Editor s Note: This Manuscript was accepted for publication June 30, This research was conducted at the Basil Hetzel Institute and Queen Elizabeth Hospital, and supported by the Hospital Research Foundation. The authors have no funding, financial relationships, or conflicts of interest to disclose. Send correspondence to Peter-John Wormald, MD, Department of Otorhinolaryngology, Head and Neck Surgery, The Queen Elizabeth Hospital, 28 Woodville Road, Woodville South, South Australia 5011, Australia. peterj.wormald@adelaide.edu.au DOI: /lary cate in biofilm form 5,10 (present in 25% 100% of CRS 11 ). CRS patients infected with S aureus experience a far more severe and often surgically resistant form of the disease than those infected with other microorganisms. 12 Treatment of CRS varies widely among clinicians, 13 but steroids (both systemic and topical) have a proven benefit in vivo. 1,14 The powerful therapeutic effect of topical steroids is thought to result from their antiinflammatory rather than bactericidal effect. 15 Steroid molecules delivered intranasally bind to the glucocorticoid receptors inside the cells of the nasal mucosa and modulate gene transcription, 16 resulting in a reduction in recruitment of inflammatory cells to the area, causing less of the erythema, edema, and pain that are associated with inflammation. However, Zeng et al. 15 showed that the in vivo effects of topical mometasone were no different than those of oral clarithromycin. Lavigne et al. 17 showed that topical budesonide instillations alone improve symptom scores in recalcitrant CRS patients. These findings suggest that steroids may have direct antibacterial effects in the sinuses, particularly against biofilms, although this conclusion was not made. This is a surprising observation considering the traditional understanding of steroid function as solely anti-inflammatory. The mechanisms underlying these observations are largely unknown. It remains uncertain if steroids 602

2 Fig. 1. (A) MBEC device. (B) Close-up view of peg immersed in well. Green denotes areas of potential biofilm growth; yellow denotes bacterial solution. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] induced a direct reduction in bacterial load, or if the observed improvement in symptoms was solely through a reduction in the host inflammatory response. Mometasone, fluticasone, and budesonide are the most commonly used intranasal steroids in CRS, 13,18 and to the best of our knowledge it has not yet been investigated whether these or any other topical steroids used in CRS have direct antibacterial or antibiofilm effects separate from their anti-inflammatory properties. This study, therefore, aimed to determine if topical steroids used in CRS reduce S aureus biofilms in vitro. Fig. 2. Z-stacks of Staphylococcus aureus biofilms taken of control pegs with a confocal scanning laser microscope. (A) SYTO 9 LIVE/DEAD BacLight stain only. (B) Propidium iodide (PI) stain only. (C) SYTO 9 and PI stains combined. Both extracellular polymeric substance matrix and cellular material are stained. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] MATERIALS AND METHODS Bacterial Strains and Culture Conditions S aureus biofilms were grown from S aureus American Type Culture Collection (ATCC) and suspended in solution as per previously documented protocols. 19,20 Biofilm Formation and Exposure to Steroids A total of 200 ll of the bacterial solution was placed in each well of a minimum biofilm eradication concentration (MBEC) device (Innovotech, Inc., Edmonton, AB, Canada) 21 (Fig. 1). Once prepared, the MBEC device was incubated at 35 C on a gyrorotary shaker (Ratek Instruments, Victoria, Australia) at 70 rpm for 48 hours. Following incubation, the biofilm-coated pegs were immersed in a new 96-well challenge plate (Greiner Bio-One, Frickenhausen, Germany). Three different steroid powders were tested; budesonide (Sigma-Aldrich, St. Louis, MO), mometasone (mometasone furoate; Sigma-Aldrich), and fluticasone (fluticasone propionate; Sigma-Aldrich). These three are the topical steroids used most commonly in CRS. 13,18 Concentration ranges were chosen around the recommended therapeutic dose for the steroids when used in CRS. Thus, the concentration tested for fluticasone and mometasone ranged from 25 lg/200 ll to 400 lg/200 ll, and from 16 lg/200 ll to 2000 lg/200 ll for budesonide. Corticosteroids have limited solubility in CSF broth, which necessitated the use of specific vehicle solvents. Consequently, fluticasone and high doses of budesonide were both dissolved in dimethyl sulfoxide (DMSO) (5 mg fluticasone in 200 ll, 50 mg budesonide in 1 ml). Lower doses of budesonide were soluble in cerebrospinal fluid (CSF) broth alone (256 lg in 200 ll). Mometasone was dissolved in chloroform (5 mg in 200 ll). The following dosing regimen was then used to encompass the clinical dose of each steroid and beyond: Fluticasone: 25 lg, 50 lg, 100 lg, 200 lg, 300 lg, 400 lg (all in 200lL of DMSO and CSF broth, recommended therapeutic dose 200 lg 13,18 ) Mometasone: 25 lg, 50 lg, 100 lg, 200 lg, 300 lg, 400 lg (all in 200 ll of chloroform and CSF broth, recommended therapeutic dose 200 lg 13,18 ) Budesonide: 16 lg, 64 lg, 128 lg, 256 lg, 500 lg, 750 lg, 1000 lg, 2000 lg (all in 200 ll of CSF broth 6 DMSO, recommended therapeutic dose 256 lg for nasal spray, 1,000lg for respules 13,18 ) Corresponding controls containing equivalent volumes of the appropriate solvent were established for each treatment. Image Acquisition and Quantification of Biofilms Following 24 hours of incubation in various control and steroid solutes (as seen in previous studies 19,20 ), pegs on which biofilm was grown were removed from the device and washed twice in 0.9% saline to remove excess planktonic bacteria, followed by immersion in 5% glutaraldehyde fixative (Sigma- Aldrich) for 45 minutes at 21 C. Biofilm pegs were washed with 0.9% saline to remove excess fixative and immersed in 1 ml of MilliQ (Millipore, Billerica, MA) water containing 1.5 ll each of both LIVE/DEAD BacLight stains SYTO9 and propidium iodide (Invitrogen Molecular Probes, Mulgrave, Victoria, Australia). This was incubated in the dark at 21 C for 15 minutes. The pegs were washed in 0.9% saline to remove excess stain and mounted on coverslips. Each specimen was scanned for biofilms using a Leica TCS SP5 confocal scanning laser microscope (CSLM) (Leica 603

3 Fig. 3. Mean and standard error of the mean biomass values (in lm 3 /lm 2 ) for fluticasone data. The highest dose of fluticasone investigated (400lg/200lL) attenuates biofilm load (denoted by the asterisk). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Fig. 4. Mean and standard error of the mean biomass values (in lm 3 /lm 2 ) for mometasone data. High doses (300lg/200lL and 400lg/200lL) of mometasone attenuate biofilm load (both denoted by the asterisks). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Microsystems, Wetzlar, Germany). CSLM is the imaging modality of choice for the identification of biofilms. 22,23 Biofilms can be easily identified on CSLM as clusters or towers of intensely fluorescing green coccus-shaped bacteria of approximately 0.5 to 2 lm in diameter 22 (Fig. 2). The Leica Application Suite Advance Fluorescence Software captured Z-stacks at two different but uniform sites on each peg. Biofilm growth is uneven across the peg, being heavy and poorly representative at the tips and menisci. 20,24 Thus, a single assessor chose the two sites of heaviest growth outside of these areas for Z-stack capture. 19,20,24 Each stack contained 120 images, 0.7 lm apart. Bacterial quantification within the biofilm (including the extracellular polymeric substance [EPS] matrix) was performed by the COMSTAT2 software programme. A single COMSTAT2 user set a threshold for all of the Z-stacks, and biomass was determined by the software. Each steroid was tested in duplicate over two separate 6-day periods, rendering a sample size of four biomass values per treatment (or control) group. This sample size was adequate to demonstrate statistically significant differences (where any existed) in previous studies using the same methodology. 19,20 P <.0001). Post hoc contrasts between dose groups (treatment vs. control) were performed to investigate these differences further (Fig. 3). Only the reduction between the treatment and control pegs at the highest concentration tested (400 lg/200 ll) was found to be statistically significant (difference lm 3 /lm 2, P 5.007). Mometasone Global comparisons revealed a statistically significant difference in the mean biomass across all six different concentration groups and their six corresponding control groups (v 2 statistic , P <.0001). Post-hoc contrasts between dose groups (treatment vs. control) were performed (Fig. 4), and significant reductions were found between the 300 lg/200 ll control and treatment (difference lm 3 /lm 2, P 5.006), and 400 lg/200 Statistical Analysis Statistical analyses were performed using SAS version 9.3 (SAS Institute Inc., Cary, NC). Data are expressed as mean and standard error of the mean biomass values for each four-value treatment or control group. Data were variable (particularly in control pegs), and so a linear generalized estimating equation (GEE) with robust variance estimation 25 was used to compare mean biomass values within each steroid group. Two-tailed P values elucidated from these tests that were <.05 were considered statistically significant. RESULTS Fluticasone Initial global comparisons within the GEE showed a statistically significant difference in the mean biomass across all six different concentration groups and their six corresponding control groups (v 2 statistic , 604 Fig. 5. Mean and standard error of the mean biomass values (in lm 3 /lm 2 ) for low-dose budesonide data. Low-dose budesonide has no effect on biofilm load. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

4 Fig. 6. Mean and standard error of the mean biomass values (in lm 3 /lm 2 ) for high-dose budesonide data showing 750 lg/200 ll, 1000 lg/200 ll, and 2000 lg/ll of budesonide attenuate biofilm load (all denoted by the asterisks). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] ll control and treatment (difference lm 3 /lm 2, P 5.034). Budesonide Global comparisons of the lower range (16 lg/200 ll to 256 lg/200 ll) showed no statistically significant differences between the six groups (v 2 statistic , P 5.112), therefore no post hoc contrasts were performed. Mean biomass values can be seen in Figure 5. Global comparisons of the higher dose range (500 lg/200 ll to 2000 lg/200 ll) showed a statistically significant difference in the mean biomass across all four different concentration groups and their four corresponding control groups (v 2 statistic , P 5.002). Post hoc testing (Fig. 6) revealed a significant reduction in biomass at the three highest concentrations tested; 750 lg/200 ll treatment versus control (difference lm 3 /lm 2, P 5.038), 1000 lg/200 ll treatment versus control (difference , P 5.009), and 2000 lg/ 200lL treatment versus control (difference lm 3 /lm 2, P 5.029). DISCUSSION This study shows that higher doses of specific steroid formulations reduce S aureus biomass in vitro. Control solutions contained equal amounts of solvents with respect to the test solutions and did not show any significant effects on established S aureus biofilms. These results indicate that the observed antibiofilm effects are mediated by the corticosteroids. The 400 lg/200 ll fluticasone group (the highest tested) showed a 96% reduction in biofilm presence. Budesonide displayed antibiofilm effects within the higher dose range investigated with 98%, 99%, and 99% reductions in biomass at 750 lg/200 ll, 1000 lg/200 ll, and 2000 lg/200 ll, respectively. The 500 lg/200ll dose of budesonide was not effective, and so it was anticipated that the lower budesonide concentrations would not affect the biofilm. Mometasone showed 65% and 98% reductions in biomass at 300 lg/200 ll and 400 lg/200 ll, respectively Our data support previous in vivo studies evaluating symptomatology in CRS patients. 15,17 Steroids currently form an important part of the treatment of most CRS patients, and their benefit has been proven. 1,13,14 Until now, the assumption has been made that steroidmediated improvement in CRS symptoms was solely due to their anti-inflammatory properties. 26 This in vitro study supports the rationale that steroids induce a positive and direct reduction in S aureus biofilm growth, and that this is independent from the inflammatory milieu. Steroids are, however, used as anti-inflammatory agents at doses much lower than those we have deemed effective in the reduction of biofilm production. This correlates somewhat with their observed clinical efficacy however; however, low-dose steroid nasal sprays do not relieve symptoms as well as high-dose steroid respules. 17,27 This has previously been attributed to the method of delivery 17,27 ; sinus intubation and instillation allows the steroids direct contact with the inflamed (and infected) mucosa. Given our data, these high doses appear to be effective at least in part because of an associated antibacterial effect. Topical steroids are not new agents in the medical management of CRS, but this study gives us new insight into their potential mechanism or mechanisms of action against bacteria; one that had previously not been considered. Increasing local steroid dosing to reduce biofilm load presents an attractive therapeutic alternative for patients resistant to antibiotic therapy. However, it is important to be mindful of the large steroid doses and prolonged exposure times used in this study, which would not necessarily be applicable or safe in vivo. It is known that solvents, including DMSO, can have bacteriostatic effects. 28 Little or no susceptibility to DMSO is seen up to a concentration of 2 vol% DMSO, 28 but incubation with 20 vol% of DMSO induces pore formation in mammalian cells large enough for small molecules (MW 40 g/mol) to enter cells freely, but no (or very small numbers) of larger molecules (MW of 630 g/mol) are able to do the same. 29 Budesonide and fluticasone have a molecular weight of over 430 g/mol. In our fluticasone and budesonide experiments, the control solutions did not show any significant effects on established S aureus biofilms even at up to 20 vol% DMSO, whereas antibiofilm effects were observed for both fluticasone and budesonide in the presence of 8 to 20 vol% DMSO. These results indicate that at concentrations up to 20 vol%, DMSO does not decrease the biomass of an established S aureus (ATCC 25923) biofilm. Techniques for the quantification of in vitro biofilms other than CSLM have been documented, namely the semiquantitative measurement of optical density of biofilm samples and staining with crystal violet. 30,31 CSLM gives a more accurate measurement of true biomass and has been used previously in similar studies, 19,20,22,23 but is certainly more technically difficult and expensive than optical density, and as such this alternative method could be used to add strength our results. 605

5 Biofilm growth on control pegs varied throughout the study. Prior published data are consistent with this observation, as biofilms grow in a stochastic manner, and even when experimental conditions are kept constant, exact structural copies are often not produced in the laboratory setting. 20,24 Nonetheless, the reduction in biofilm growth in the pegs exposed to higher steroid concentrations relative to their control counterparts was consistent and statistically significant. The strength of this study was the ability to assess directly the presence (or absence) of any direct antibacterial effect of topical steroids commonly used in the treatment of CRS. The protocol eliminates possible antiinflammatory effects, which would be indistinguishable from any antibacterial effect in vivo. Currently one of the mainstays of managing recalcitrant CRS patients is the use of topical therapies. 13 The method of delivery is usually via a 240 ml squeeze bottle. When this method is used after sinus surgery, there is good drug penetration of the sinuses. 32 Topical steroids delivered to the sinuses via a squeeze bottle reduce the rate of bacteria cultured from the sinuses. 33 This adds evidence and supports the findings of this study that topical steroids (in high doses) may have a direct deleterious effect on the bacteria of the sinuses. Although this study assesses only the in vitro effects of steroids on S aureus biofilms, it warrants mention that indirect antibacterial effects of corticosteroids are likely in vivo via a reduction in the viability and activation of eosinophils. 34,35 The mechanism of action of glucocorticoids on S aureus biofilms is not known. To the best of our knowledge, no glucocorticoid receptors have been identified in bacterial cells. Bacteria do not have a nucleus, and transcription and translation is coupled and occurs in the bacterial cytoplasm. 36 If it can be shown that the glucocorticoids used exert their antibacterial effects upon entering the bacterial cytoplasm, it could be hypothesized that they bind bacterial DNA, RNA, and/or transcription factors present within the cytoplasm, thereby directly or indirectly affecting gene transcription. Newly developed force- and fluorescence-based single-molecule methods could help in the elucidation of the molecular events affected. 36 Alternatively, if glucocorticoids can be demonstrated to be localized into the bacterial cell membrane, they could affect membrane fluidity and dynamics by changing the content and function of bacterial cell membrane molecules. In addition to their possible direct modes of action, corticosteroids could exert their effects in an indirect way by destabilizing the biofilm matrix. Further experiments studying the localization of the corticosteroids with respect to the bacterial cells and/or within the bacterial cells may help to further elucidate the mode of action of corticosteroids on S aureus biofilms. This study poses a number of new questions regarding steroids and their use in CRS. The relative magnitude of steroidal anti-inflammatory and direct antibiofilm effects in vivo is unknown, as is the mechanism of action of their biofilm-reducing effect. The exposure of the biofilms in this study to steroids for 48 hours does not reflect the true exposure of the sinuses to intranasal corticosteroid preparations; however, 48 hours of exposure is what has been used in previous studies with the same methodology. 19,20 The aim of our study was to investigate the direct in vitro effects of the steroids; the application of these results in vivo is beyond the scope of this research, but is certainly an area of great interest. Further studies to determine the effect of topical steroids on other biofilm species need to be performed and correlated with improved patient outcomes. Further studies are also required to find a safe and effective steroid dose in vivo to optimize biofilm-reducing and antiinflammatory actions, while minimizing the side effect profile, improving the overall outcome and treatment of CRS patients. CONCLUSION In conclusion, commonly used intranasal steroids directly limit the growth of in vitro S aureus biofilms in a concentration-dependent manner. ACKNOWLEDGMENTS The authors thank Thomas Sullivan of the University of Adelaide, the Basil Hetzel Institute, the Hospital Research Foundation, and Adelaide Microscopy. BIBLIOGRAPHY 1. Snidvongs K, Kalish L, Sacks R, Craig JC, Harvey RJ. Topical steroid for chronic rhinosinusitis without polyps. Cochrane Database Syst Rev 2011;(8):CD Ahovuo-Saloranta A, Borisenko OV, Kovanen N, et al. Antibiotics for acute maxillary sinusitis. 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