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

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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
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/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

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
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).

9
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 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)

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
    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.

18
Results
19
Multiple Strains of each Bacteria form Biofilm on
Lotrafilcon A Lenses


p 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 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. 2672-84
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
  • 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
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