Title: 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
2Disclosures
- 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
3Acanthamoeba (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/disinfectant
s, 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
4Microbial adherence to contact lenses has long
been recorded
5Bacterial Adhesion Studies
6Biofilms
- 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)
7Biofilms can form in vivo
8Phases of Biofilm Development
0 h, Adhesion
8 h, Proliferation
11 h, Micro-colonies
48 h, Cells within matrix
- Green fluorescence - ConA binding to
polysaccharides - Red fluorescence FUN1 staining of
metabolically active cells
- Chandra et al. (2001) J. Bacteriology 183 (18).
9Schematic Representation of Biofilm Development
in C. albicans
Top view Side view
10Specific 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.
11METHODS
- 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
12Organisms 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
13Contact 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
14Methods
- Growth Conditions
- Bacteria were grown overnight at 37C 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 37C 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)
15Methods
- 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).
16Methods
- 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.
17Methods
- 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
manufacturers 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.
18Results
19Multiple Strains of each Bacteria form Biofilm on
Lotrafilcon A Lenses
p
20Ultrastructural/ scanning EM analysis of
Bacterial Biofilms formed on Lotrafilcon A lenses
21Confocal 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.
22Contact Lens Solutions are Active Against
Planktonic but not Biofilm Forms of Bacteria
23 P
24SUMMARY
- 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.
25SUMMARY
- 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.
26Acanthamoeba 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. 2672-84
27Variables 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
28Steps 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
29Optimization 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
30Real World Number of Coagulase-negative
Staphylococcus CFUs Isolated From Worn
Lotrafilcon A Lenses
Unpublished data courtesy of Dr. Loretta
Szczotka-Flynn
31Mean and Max CFUs Across All Visits
Unpublished data courtesy of Dr. Loretta
Szczotka-Flynn
32Acknowledgments
- Collaborators
- Loretta Szczotka-Flynn OD, MS
- Yoshi Imamura, PhD
- Changping Yu, PhD
- Jyotsna Chandra, PhD
- Pranab K. Mukherjee, PhD
- Eric Pearlman, PhD
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