A Method for Testing Activity of Contact Lens Disinfectants Against Acanthamoeba: The Real World

1. A Method for Testing Activity of Contact Lens Disinfectants Against Acanthamoeba: The Real World Mahmoud Ghannoum, Ph.D. Director & Professor Center for Medical Mycology Department of Dermatology Case Western Reserve University Cleveland, OH (USA) Microbiological Testing for Contact Lens Care Products Workshop Jan 22-23, 2009

2. Disclosures • Received grants, acted as member of advisory board, and/or speaker of the following companies: • Pfizer, Merck, Schering-Plough, Johnson & Johnson, Stiefel, NovaBay, Great Lakes Pharmaceuticals, Alcon (grant pending) • Expert witness on biofilms

3. Acanthamoeba (AM) and Biofilms In vitro studies have shown that Gram- bacteria may act as a preferred food source for AM Bacterial biofilms provides a suitable environment for the growth of AM Little is known about the survival of AM in biofilms However, it is likely that biofilms afford protection for AM from antimicrobials/disinfectants, as is the case for bacteria. Therefore, any method developed to assess the efficacy of contact lens disinfectants against AM should incorporate a bacterial biofilm

4. Microbial adherence to contact lenses has long been recorded

5. Bacterial Adhesion Studies

6. Biofilms • Structural community of microorganisms adherent to biotic/abiotic surface. • Encased in a self-produced extracellular matrix (ECM) • Resistant to antimicrobial agents • Can result in both invasive and non-invasive infections Bacterial mats, Grand Prismatic Spring, Yellowstone National Park (Google Images)

7. Biofilms can form in vivo

8. Phases of Biofilm Development 0 h, Adhesion 8 h, Proliferation Chandra et al. (2001) J. Bacteriology 183 (18). 11 h, Micro-colonies 48 h, Cells within matrix • Green fluorescence - ConA binding to polysaccharides • Red fluorescence – FUN1 staining of metabolically active cells

9. EARLY INTERMEDIATE MATURE Schematic Representation of Biofilm Development in C. albicans Top view Side view

10. Specific Aims • To determine whether 3 different bacterial strains commonly associated with contact lens keratitis and inflammation can form biofilm on a silicone hydrogel contact lens. • To assess the antimicrobial activities of contact lens care solutions against bacterial cells grown under planktonic or biofilm conditions.

11. METHODS • Ability of strains to form biofilm • Time course of biofilm formation • Determined by monitoring biofilm development at different time points • Scanning electron microscopy (SEM) analysis of biofilms formed on contact lenses • Confocal scanning laser microscopy (CSLM) analyses of biofilm architecture and thickness • Evaluation of Antibacterial Activity of Contact Lens Care Solutions

12. Organisms Tested • These species are common causative agents of contact lens associated infections and inflammation • §The LASH study (LSF, University Hospitals Case Medical Center) is an ongoing prospective cohort study of 208 users of lotrafilcon A lenses worn continuously for up to 30 days. Szczotka-Flynn et al. (Submitted) Cornea

13. Contact lens care solutions tested • Five most common multipurpose solutions (MPSs) or multipurpose disinfecting solutions (MPDSs), and a hydrogen peroxide based care system, were tested Szczotka-Flynn et al. (Submitted) Cornea

14. Methods • Growth Conditions • Bacteria were grown overnight at 37°C in tryptic soy broth (TSB) washed 3 times with phosphate buffered saline (PBS) • Biofilm formation • Lenses were washed with PBS, placed in 12-well tissue culture plates with 4 ml standardized cell suspensions (O.D. 660 nm = 0.1), and incubated for 120 min at 37 °C (adherence phase) • Non-adherent cells were removed by gentle washing, and incubated at 37°C on a rocker • Biofilms formed on lenses were washed with PBS, sonicated, and vortexed • The resulting cell suspension was serially diluted and quantitatively cultured on Mueller-Hinton (MH) agar for determination of colony forming units (CFUs)

15. Methods • Scanning electron microscopy (SEM) analysis of biofilms formed on contact lenses • Confocal scanning laser microscopy (CSLM) analyses of biofilm architecture and thickness • Biofilms grown on contact lenses were transferred to 12-well plates and stained with the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Eugene, OR).

16. Methods • Evaluation of Activity of Contact Lens Care Solutions Against Bacterial Biofilms • Lotrafilcon A lenses with biofilm were washed by PBS (for at least five seconds) to simulate the rinsing step • Lens was put in 4 ml of one of the indicated contact lens care solutions in 12-well plates and incubated at room temperature according to manufacturing company recommendations. • 4 h (ReNu MultiPlus and MoistureLoc, AQuify, COMPLETE MoisturePlus) • 6 h (OPTI-Free, Clear Care) • Clear Care • Lenses with biofilm were put into the lens case supplied by the manufacturing company using their recommended amount of solution because this solution • After treatment, lenses were washed by PBS as above, transferred to 1.5 ml tube with 1.0 ml of PBS, sonicated for 5 min and agitated by vortex for 3 min. • Cell suspensions were treated with Dey-Engley Neutralizing Broth (DEB, Difco Laboratories) for 15 min and serial dilutions were spread on Tryptic Soy Agar (TSA) plates to evaluate viability. • Each strain was tested three independent times.

17. Methods • Evaluation of Antibacterial Activity of Contact Lens Care Solutions Against Planktonic Bacteria • International Organization for Standardization (ISO 14729) Stand Alone Procedure guidelines. • Absorbances of cell suspensions were adjusted to obtain 4.0 x 107 cfu/ml. • 0.1 ml suspension of each strain was mixed with 10 ml of each lens care solution and incubated per manufacturer recommended times at room temperature: • 4 h (ReNu MultiPlus and MoistureLoc, AQuify, and COMPLETE MoisturePlus) • 6 h (for OPTI-Free and Clear Care) • Clear Care • Cell suspensions were placed in the manufacturer’s platinum coated disk container with their specified volume of solution so that the neutralization step was effectively accomplished. • After treatment, the resultant mixture was treated with DEB and serial dilutions were spread on TSA plates for CFU counting. • Each strain was tested three independent times.

18. Results

19. Multiple Strains of each Bacteria form Biofilm on Lotrafilcon A Lenses * * *p<0.05

20. Ultrastructural/ scanning EM analysis of Bacterial Biofilms formed on Lotrafilcon A lenses

21. Confocal analysis of the architecture of biofilms formed by P. aeruginosa, S. marcescens and S. aureus. Panels show orthogonal view of biofilms formed on silicone hydrogel contact lens by (A) P. aeruginosa, (B) S. marcescens, or (C) S. aureus. Magnification, x40.

22. Contact Lens Solutions are Active Against Planktonic but not Biofilm Forms of Bacteria

23. * * * * * * * * P<0.05 compared to untreated control

24. SUMMARY • We report a reproducible in vitro model of bacterial biofilm formation on unworn lotrafilcon A silicone hydrogel contact lenses • P. aeruginosa, S. marcescens, and S. aureus can form biofilms on this contact lens polymer. • Although lens care solutions are effective against planktonic bacterial growth, overall they are much less effective against bacterial biofilms.

25. SUMMARY • Inadequacies exist in the testing procedures recommended by the FDA Premarket Notification 510(k) Guidance Document for Contact Lens Care Products. • Currently, the disinfecting effect of contact lens care solutions for licensing purposes continues to be tested against planktonically grown microbial cells. • Biofilms are associated with contact lenses, their carrier cases, and adverse events. • We recommend the testing of lens care solutions for activity against biofilms prior to launching a new product.

26. Acanthamoeba vs. Contact Lens Care Solutions: Test Method Variables • Standard method for working with Acanthamoeba has not been established • Therefore, contradictory result for activity against Acanthamoeba may be due to variations in the techniques used for evaluating contact lens care solutions • Variables affecting results include organism tested, growth form (trophozoites or cysts), inoculum preparation and size, assay and neutralization, quantitation and viability Buck et al. 2000 CLAO J. 26:72-84

27. Variables Noted in Several Test Methods Employed for Assessment of the Efficacy of Contact Lens Disinfectants Against Acanthamoeba Anger & Lally (2008) Eye Contact Lens 34(5): 247-53

28. Steps Involved in Development of a Standard Method to Evaluate the Efficacy of Lens Disinfectants against AM • Establishment of a standard method to evaluate activity of contact lens disinfectants against Acanthamoeba is critical • Steps involved in the development of a standard method are: • Optimize the variables • Studies to determine intra- and inter-laboratory agreement using the optimized method • Identify QC isolates that should be used as reference strains

29. Optimization of Variables • Biofilm formation • Which organism – Pseudomonas, Xanthomonas, Enterobacter, other? • What strain – ATCC or clinical • Inoculum size – real world vs. in vitro • Biofilm phase – early vs. mature • Acanthamoeba growth • Which form – trophozoite or cyst • Inoculum size • Incubation time with biofilms

30. Real World: Number of Coagulase-negative Staphylococcus CFUs Isolated From Worn Lotrafilcon A Lenses Unpublished data courtesy of Dr. Loretta Szczotka-Flynn

31. Mean and Max CFUs Across All Visits Unpublished data courtesy of Dr. Loretta Szczotka-Flynn

32. Acknowledgments • Funding Support: • NIH • R01 DE017486-01A1, R01DE 13932-4 (MG) • K23 EY015270-01 (LSF) • EY14362 (EP) • P30 EY11373 (EP) • Bristol Myers Squibb Freedom to Discover Award (MG) • American Heart Association (Scientist Development Grant 0335313N) Award (PKM) • Research to Prevent Blindness Foundation • Ohio Lions Eye Research Foundation Collaborators: Loretta Szczotka-Flynn OD, MS Yoshi Imamura, PhD Changping Yu, PhD Jyotsna Chandra, PhD Pranab K. Mukherjee, PhD Eric Pearlman, PhD